Methods for the synthesis of monofucosylated oligosaccharides terminating in di-N-acetyllactosaminyl structures

ABSTRACT

Disclosed are methods for the preparation of monofucosylated and sialylated derivatives of the compound βGal(1-4)βGlcNAc(1-3)βGal(1-4)βGlcNAc-OR. In particular, the methods of this invention provide for a multi-step synthesis wherein selective monofucosylation is accomplished on the 3-hydroxy group on only one of the GlcNAc units found in the βGal(1-4) βGlcNAc (1-3) βGal (1-4) βGlcNAc-OR compound. In this step, monofucosylation is achieved by use of the α(1-3)fucosyltransferase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/889,017filed May 26, 1992 which, in turn, is a continuation-in-part of U.S.Ser. No. 07/771,259 filed Oct. 2, 1991, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/714,161 filed Jun. 10, 1991,each of these applications is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to methods for the preparation ofmonofucosylated and sialylated derivatives of the compoundβGal(1-4)βGlcNAc-(1-3)βGal(1-4)βGlcNAc-OR. In particular, the methods ofthis invention provide for a multi-step synthesis wherein selectivemonofucosylation is accomplished on the 3-hydroxy group on only one ofthe GlcNAc units found in the βGal(1-4)βGlcNAc-(1-3)βGal(1-4)βGlcNAc-ORcompound. In these methods, monofucosylation is achieved by the use ofan α(1-3)fucosyltransferase. This invention is also directed tocompounds prepared by the herein described methods.

2. References

The following references are cited in this application as superscriptnumbers at the relevant portion of the application:

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All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

3. State of the Art

The art teaches that specific oligosaccharides such as sialylated andfucosylated structures are involved as ligands in cell adhesionphenomena.^(1a-1c) Similarly, oligosaccharide glycosides relating toblood group determinant structures have been found to impartimmunosuppressive and tolerogenic properties to mammals when the mammalswere previously challenged with an antigen. See Ippolito et al.²², whichapplication is incorporated herein by reference in its entirety. In thisregard, oligosaccharide glycosides relating to blood group determinantstructures include the compound βGal(1-4)βGlcNAc(1-3)βGal(1-4)βGlcNAc-ORdepicted in FIG. 1 of this application as compound 1a.

Ippolito et al.²² further discloses that blood group determinantoligosaccharide glycosides having a sialic acid group (or an analoguethereof) at the non-reducing sugar terminus of the oligosaccharideglycosides and which are also monofucosylated possess immunosuppressiveand tolerogenic properties (e.g., sialyl Lewis^(X) --Compound III inFIG. 12 of Ippolito et al.²²).

In view of the above, we desired to prepare a monofucosylated derivativeof compound 1a having a sialic acid group (or an analogue thereof) atthe non-reducing sugar terminus of this compound wherein the fucosylgroup was pendant to the 3-hydroxy of only one of the GlcNAc groups.

A synthetic approach employing enzymatic sialylation and fucosylationsteps is particularly appropriate in order to provide an efficient routefor the preparation of sialylated and monofucosylated derivatives ofcompound 1a. In this regard, since the work of Sabesan et al.,²-sialyltransferases, mostly the βGal(1-4)βGlcNAc α(2-6)- and theβGal(13/4)βGlcNAc α(2-3)-sialyltransferases from rat liver and theβGal(1-3)βGalNAc α(2-3)sialyltransferase from porcine submaxillary glandhave often been used for synthetic purposes.³ The former twosialyltransferases are useful in sialylating a terminalβGal(1-4)βGlcNAc- group in an oligosaccharide glycoside. The lattersialyltransferase which has a wide acceptor specificity, is useful insialylating a terminal βGal(1-3)βGlcNAc-group in oligosaccharideglycosides based on the Lewis^(c) (Type I) backbone⁴ but, because of thelow affinity of this enzyme for the Type II backbone, the synthesis ofsialylated N-acetyllactosaminyl structures, such as those present in thesialyl Lewis^(x),⁵ sialyl dimeric Lewis^(x6) or the correspondinginternally monofucosylated derivative.sup. 7, by use of thissialyltransferase is much more difficult.⁴

By using fucosyltransferases of various specificities, the biosyntheticpathway leading to sialyl Lewis^(x) and the sialylated dimeric Lewis^(x)structures has been shown to proceed by the sequential sialylationfollowed by fucosylation of the Type II precursors.^(8a-d) A similarprocess "extension, sialylation, fucosylation" has also been proposed⁹to lead to internally fucosylated repetitive Type II terminalstructures, such as: αNeu5Ac(2-3)βGal(1-4)βGlcNAc(1-3)βGal(1-4)-[αFuc(1-3)]βGlcNAc-.⁹ The identification of the new terminal structureβGal(1-4)βGlcNAc(1-3)βGal(1-4)-[[αFuc(1-3)]βGlcNAc-, defined by theantibody ACFH-18¹⁰, led to a proposed new biosynthetic pathway such as"elongation followed by selective internal fucosylation". While patternsof initial internal monofucosylation of di-N-acetyllactosaminylglycolipids have been observed for fucosyltransferases present in LECIIChinese Hamster Ovary mutant¹¹ and in human colonic adenocarcinoma Colo205 cells^(8b), these fucosyltransferases are not readily availableand/or do not selectively lead to monofucosylated structures. Similarly,while fucosyltransferases possessing the specificity required for thesynthesis of the internally fucosylated structureαNeu5Ac(2-3)βGal(1-4)GlcNAc-βGal(1-4)[αFuc(1-3)]GlcNAc have beenidentified³⁴ and in one case a recombinant enzyme³⁵ has been identified,their availability is also limited. Moreover, otherα(1-3)fucosyltransferases do not transfer L-fucose ontoN-acetylglucosamine moieties found in acceptors possessing a terminalαNeu5Ac(2-3)βGal(1-4 )βGlcNAc- sequence.¹²,13,34 It has already beennoted that "the order of addition of α(1-3) fucose inN-acetyllactosaminyl sequences of glycoconjugates will then depend uponthe particular α(1-3)fucosyltransferase present"¹¹.

The single fucosylation at the internal N-acetylglucosamine unit of theα(2-6)sialyl di-N-acetyllactosaminyl sequence leading to the terminalstructureαNeu5Ac(2-6)βGal(1-4)βGlcNAc(1-3)-Gal(1-4)-[αFuc(1-3)]βGlcNAc-^(8a)(also proposed for a sialylfucopentaose from human milk¹⁴) is inagreement with the proposed mutually exclusive glycosylation pattern ofthe βGal(1-4)βGlcNAc α(2-6)sialyltransferase and the βGal(13/4)βGlcNAcα(13/4)fucosyltransferase in the synthesis of asparaginyl linkedoligosaccharides in glycoproteins.¹⁵

In view of the above, processes which would enzymatically preparesialylated and monofucosylated derivatives of compound 1a without theneed to employ a fucosyltransferase specific for monofucosylation wouldbe particularly desirable.

The present invention is based, in part, on the discovery of syntheticpathways which utilizes enzymatic fucosylation and sialylation steps andwhich result in the selective formation of monofucosylated derivativesof compound 1a without the need to employ a fucosyltransferase which isspecific for monofucosylation on either of the GlcNAc units of compound1a.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the discovery that fucosylationonto the 3-hydroxyl group of the GlcNAc saccharide in a βGal(1-4)βGlcNAcdisaccharide via an α(1→3)fucosyltransferase (e.g., βGal(13/4)βGlcNAcα(13/4) fucosyltransferase) is dependent on the presence of a 6-hydroxylgroup on the Gal saccharide and when this hydroxyl group is blocked by aremovable blocking group, fucosylation on the neighboring GlcNAc groupis prevented. In this aspect, the methods of this invention employ thischaracteristic of α(1-3)fucosyltransferases to provide for a means toselectively monofucosylate compound 1a which are used advantageously toprepare compounds 5a and 12.

In another method aspect, the present invention is directed to thediscovery of enzymatic methods and chemical/enzymatic methods to preparethe compoundαNeu5Ac(2-3)βGal(1-4)βGlcNAc(1-3)βGal(1-4)-[αFuc(1-3)]βGlcNAc-OR.

Thus, in one of its method aspects, the present invention is directed toa method for preparation of a compound of the formula I: ##STR1##wherein R is hydrogen, a saccharide or an oligosaccharide, or an aglycongroup having at least one carbon atom, Y is L-fucose or a compatibleanalogue of L-fucose, and Z is sialic acid or a compatible analogue ofsialic acid, which method comprises the following steps:

(a) preparing a compound of the formula II ##STR2## wherein R is asdefined above and X is a removable blocking group;

(b) fucosylating the compound prepared in (a) above with anα(1-3)fucosyltransferase so as to form a monofucosylated derivative ofthe formula III: ##STR3## wherein X, Y and R are as defined above;

(c) removing the removable blocking group from the compound formed in(b) above; and

(d) sialylating the compound formed in (c) above with sialic acid or acompatible analogue of sialic acid using an α(2-3)sialyltransferase soas to form the compound of formula I.

In regard to the above, the sialylation of the oligosaccharide glycosideso as to form an α(2-3)sialyl residue at the non-reducing sugar terminusof the oligosaccharide glycoside is necessarily after removing theblocking group because sialylation with an α(2-3)sialyltransferaserequires the presence of a free hydroxyl group at the 6-position of theterminal galactose residue on the oligosaccharide glycoside.

In another of its method aspects, the present invention is directed to amethod for preparation of a compound of the formula IV: ##STR4## whereinR is a hydrogen, a saccharide or an oligosaccharide, or an aglycon grouphaving at least one carbon atom, Y' is L-fucose or a compatible analogueof L-fucose and Z is sialic acid or a compatible analogue of sialicacid, which method comprises the following steps:

(a) preparing a compound of the formula V ##STR5## wherein R is asdefined above and X' is a removable blocking group;

(b) sialylating the compound formed in (a) above with sialic acid or acompatible analogue of sialic acid using an α(2-3)sialyltransferase;

(c) fucosylating the compound prepared in (a) above with anα(1-3)fucosyltransferase so as to form a monofucosylated derivative ofthe formula VI: ##STR6## wherein X', Y' and R are as defined above; and

(d) removing the removable blocking group from the compound formed in(c) above so as to form a compound of formula IV

with the proviso that X' is a blocking group other than sialic acid.

In regard to the above, the sialylation of the oligosaccharide glycosideso as to form an α(2-3)sialyl residue at the non-reducing sugar terminusof the oligosaccharide glycoside is necessarily before the fucosylationstep because sialylation with an α(2-3)sialyltransferase will notproceed when Y' is L-fucose or a compatible analogue of L-fucose.

Preferred removable blocking groups for use in the above describedmethods include sialic acid groups and benzyl groups and any other groupthat can be introduced either enzymatically or chemically on theprecursor leading to II or V and later selectively enzymatically orchemically removed in mild conditions compatible with the nature of theproduct. In compound V, the blocking group X' is not sialic acid becausethis compound would be difficult to synthesize.

In one of its composition aspects, the present invention is directed toa compound of the formula VII: ##STR7## wherein R is hydrogen, asaccharide or an oligosaccharide, or an aglycon having at least 1 carbonatom, Y and Y' are selected from the group consisting of hydrogen,L-fucosyl and a compatible analogue of L-fucose with the proviso thatone of Y and Y', but not both, is hydrogen, and Z is sialic acid or acompatible analogue of sialic acid.

In a preferred embodiment, the aglycon moiety, R, is selected from thegroup consisting of --(A)--Z' wherein A represents a bond, an alkylenegroup of from 2 to 10 carbon atoms, and a moiety of the formula --(CH₂--CR₂ G)_(n) -- wherein n is an integer equal to 1 to 5; R₂ is selectedfrom the group consisting of hydrogen, methyl, or ethyl; and G isselected from the group consisting of hydrogen, halogen, oxygen,sulphur, nitrogen, phenyl and phenyl substituted with 1 to 3substituents selected from the group consisting of amine, hydroxyl,halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbonatoms; and Z' is selected from the group consisting of hydrogen, methyl,phenyl, nitrophenyl and, when G is not oxygen, sulphur or nitrogen and Ais not a bond, then Z' is also selected from the group consisting of--OH, --SH, --NH₂, --NHR₃, --N(R₃).sub. 2, --C(O)OH, --C(O)OR₃,--C(O)NH--NH₂, --C(O)NH₂, --C(O)NHR₃, --C(O)N (R₃)₂, and --OR₄ whereineach R₃ is independently alkyl of from 1 to 4 carbon atoms and R₄ is analkenyl group of from 3 to 10 carbon atoms with the proviso that when Ais a bond then Z' is not hydrogen.

Preferably, the aglycon group is a hydrophobic group. Most preferably,the aglycon moiety is a hydrophobic group selected from the groupconsisting of --(CH₂)₈ COOCH₃ and --(CH₂)₅ OCH₂ CH═CH₂ and --(CH₂)₈ CH₂OH.

The monosialylated and monofucosylated compounds of this invention areparticularly useful in modulating a cell-mediated immune inflammatoryresponse. Accordingly, in another of its composition aspects, thepresent invention is directed to a pharmaceutical composition suitablefor administration to a mammal (e.g., human) which comprises apharmaceutically inert carrier and an effective amount of the compoundof Formula I or IV to modulate a cell-mediated immune response in saidmammal.

In another of its method aspects, the present invention is directed to amethod for modulating a cell-mediated immune response in a mammal whichmethod comprises administering to said mammal an amount of a compound ofFormula I or IV effective in modulating said immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the synthetic pathway leading to Sialyl dimericLewis^(x) and internally monofucosylated derivatives thereof. In FIG. 1,the nomenclature for compound 1a is βGal(1-4)βGlcNAc(1-3)βGal(1-4)βGlcNAc-OR, sometimes called di-N-acetyllactosaminyltetrasaccharide. Similarly, the hexasaccharide moiety present incompounds 5a and 5b in FIG. 1 is sometimes called VIM-2 epitope orCD-65⁵ and 7a and 7b are called sialyl dimeric Lewis^(x).

FIG. 2 illustrates the synthetic pathway leading to the externallymonofucosylated derivatives of the sialyl di-N-acetyllactosaminylhapten.

FIG. 3 illustrates an enzymatic pathway leading to monofucosylated andmonosialylated compounds of Formula I.

FIG. 4 illustrates an alternative chemical synthesis of trisaccharide 19which can then be used as per FIG. 3 to prepare monofucosylated andmonosialylated compounds of Formula I.

FIG. 5 illustrates that the enzymatic pathway set forth in FIG. 3 can beused to extend the structure of the hexasaccharides of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, in part, to the discovery thatselective monofucosylation of compound 1a (i.e.,βGal(1-4)βGlcNAc(1-3)βGal-(1-4)βGlcNAc-OR), can be achieved byappropriately blocking the 6-hydroxy group on the galactose unitadjacent to GlcNAc unit (in the non-reducing sugar direction) so as toprevent fucosylation of the GlcNAc unit.

The present invention is also directed, in part, to the discovery thatthe preparation of compounds of Formula I can be achieved by a completeenzymatic process or by a chemo/enzymatic process.

In either case, the synthetic steps employed in the synthesis of themonofucosylated derivatives are critical to produce the monofucosylatedderivatives.

The present invention is still further directed to the discovery thatthe compounds of Formula I and IV are useful for in vivo modulation of acell mediated immune response in mammals, including humans.

However, prior to discussing this invention in further detail, thefollowing terms will first be defined.

A. Definitions

As used herein, the following terms have the definitions given below:

The term "cell-mediated immune response to an antigen in a mammal"refers to those mammalian immune responses which are mediated bycell-cell interactions. Included within this term are cell mediatedinflammatory responses to an antigen such as DTH responses as well ascell-mediated inflammatory responses arising from myocardial infarction,virus-induced pneumonia, shock and sequelae (e.g., multiple organfailure), adult respiratory distress syndrome, psoriasis, arthritis, andthe like. Preferably, the cell-mediated immune response is aleucocyte-mediated response.

The term "N-acetyllactosamine" or "LacNAc" refers to the disaccharideβGal(1→4)βGlcNAc which is represented by the formula: ##STR8##

The term "di-N-acetyllactosaminyl structures" means that oneN-acetyllactosamine unit is glycosidically linked in a β linkage to the3-OH of the βGal of the second unit attached to the aglycon.

The term "sialic acid" means all of the naturally occurring structuresof sialic acid including (N-acetylated)5-amino-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonic acid("NeuSAc") and the naturally occurring analogues of Neu5Ac, includingN-glycolyl neuraminic acid (Neu5Gc) and 9-O-acetyl neuraminic acid(Neu5,9Ac₂), which are compatible with the selected sialyltransferase. Acomplete list of naturally occurring sialic acids known to date areprovided by Schauer²³.

Naturally occurring sialic acids which are recognized by a particularsialyltransferase so as to bind to the enzyme and are then available fortransfer to an appropriate acceptor oligosaccharide structure are saidto be compatible with the sialyltransferase and are sometimes referredto herein as a "compatible naturally occurring sialic acid".

The term "analogues of sialic acid" refers to analogues of naturallyoccurring structures of sialic acid including those wherein the sialicacid unit has been chemically modified so as to introduce and/or removeone or more functionalities from such structures. For example, suchmodification can result in the removal of an --OH functionality, theintroduction of an amine functionality, the introduction of a halofunctionality, and the like.

Certain analogues of sialic acid are known in the art and include, byway of example, 9-azido-Neu5Ac, 9-amino-Neu5Ac, 9-deoxy-Neu5Ac,9-fluoro-Neu5Ac, 9-bromo-Neu5Ac, 8-deoxy-Neu5Ac, 8-epi-Neu5Ac,7-deoxy-Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac,4-N-acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, 3-hydroxy-Neu5Ac,3-fluoro-Neu5Ac acid as well as the 6-thio analogues of Neu5Ac. Thenomenclature employed herein in describing analogues of sialic acid isas set forth by Reuter et al.²⁴

Insofar as sialyltransferases are designed to transfer or donatecompatible naturally occurring sialic acids, analogues of Neu5Ac aresometimes referred to herein as "artificial donors" whereas thecompatible naturally occurring sialic acids are sometimes referred toherein as the "natural donors".

The term "oligosaccharide" as used in conjunction with the Rsubstituent, refers to a carbohydrate structure having from 2 to about10 saccharide units. The particular saccharide units employed are notcritical and include, by way of example, all natural and syntheticderivatives of glucose, galactose, N-acetylglucosamine,N-acetyl-galactosamine, fucose, sialic acid, 3-deoxy-D,L-octulosonicacid, and the like.

In addition to being in their pyranose form, all sugars recited hereinare in their D form except for fucose which is in its L form.

The term "sialyltransferase" refers to those enzymes which transfer acompatible naturally occurring sialic acid, activated as its cytidinemonophosphate (CMP) derivative, to the terminal galactose group presenton natural acceptor structures comprising those terminating inβGal(1-4)βGlcNAc and βGal(1-3)βGlcNAc disaccharides and include enzymesproduced from microorganisms genetically modified so as to incorporateand express all or part of the sialyltransferase gene obtained fromanother source, including mammalian sources.

Such sialyltransferases comprise those that have been identified in theliterature as leading to the following structures:

    αNeu5Ac(2-3)βGal(13/4)βGlcNAc-.sup.25

    αNeu5Ac(2-6)βGal(1-4)βGlcNAc-.sup.25,26

Analogues of sialic acid which are recognized by a particularsialyltransferase so as to bind to the enzyme and are then available fortransfer to an appropriate acceptor oligosaccharide structure are saidto be compatible with the sialyltransferase and are sometimes referredto herein as a "compatible analogue of sialic acid". Because thetransfer reaction employs a sialyltransferase, it goes without sayingthat an analogue of sialic acid employed in such a reaction must be acompatible analogue of sialic acid.

CMP-nucleotide derivative of Neu5Ac refers to the compound: ##STR9##CMP-derivatives of analogues of sialic acid refer to those compoundshaving structures similar to that above with the exception that theNeu5Ac residue is replaced with an analogue of sialic acid.

The term "α(1-3)fucosyltransferase" refers to any fucosyltransferasewhich transfers L-fucose and compatible analogues of L-fucose fromGDP-fucose to the 3 hydroxy position of GlcNAc in a LacNAc group(βGal(1-4)βGlcNAc) in an oligosaccharide glycoside and which does notdiscriminate between βGal(1-4)βGlcNAc groups in the oligosaccharideglycoside. The particular α(1-3)fucosyltransferase employed iscompatible with the intended reaction. That is to say that the selectedα(1-3) fucosyltransferase will bind to the oligosaccharide glycosideemployed and transfer L-fucose to the 3 hydroxy position of GlcNAc in aβGal(1-4)βGlcNAc group of the oligosaccharide glycoside. Suitablefucosyltransferases include the known βGal(1→3/4)βGlcNAcα(1→3/4)fucosyltransferase which is readily obtained from humanmilk⁴,70,72 and the βGal(1→4)βGlcNAc α(1→3)fucosyltransferase which isalso found in human serum and is co-recovered with theβGal(1→3/4)βGlcNAc α(1→3/4)fucosyltransferase. A recombinant form ofβGal(1→3/4)βGlcNAc α(1→3/4)fucosyltransferase is also available⁶⁸,69.

Compatible analogues of L-fucose refer to naturally occurring andsynthetic analogues of fucose including those where the fucose unit hasbeen chemically modified so as to introduce and/or remove one or morefunctionalities from this structure. For example, such modification canresult in the removal of an --OH functionality, the introduction of anamine functionality, the introduction of a halo functionality, and thelike.

Certain compatible analogues of fucose are known in the art and include,by way of example, 3-deoxy-fucose, arabinose, and the like.¹⁸

The term "removable blocking group" refers to any group which when boundto the 6-hydroxyl of the galactose unit in a βGal(1-4)βGlcNAc groupprevents fucosylation of the 3-hydroxyl of the GlcNAc by anα(1-3)fucosyltransferase and which group can be removed by conventionalchemical or enzymatic steps to reestablish the 6-hydroxyl on thegalactose unit. The particular removable blocking group employed is notcritical and preferred removable blocking groups include Neu5Ac andbenzyl substituents and any other group that can be introduced eitherenzymatically or chemically on the precursor leading to II or V andlater selectively enzymatically or chemically removed in mild conditionscompatible with the nature of the product. One such additionalcontemplated blocking group is α-galactose which can be removedenzymatically with an α-galactosidase.

The term "removable protecting group" refers to any group which whenbound to one or more hydroxyl groups of the galactose,N-acetylglucosamine, etc. which prevent reactions from occurring atthese hydroxyl groups and which protecting group can be removed byconventional chemical or enzymatic steps to reestablish the hydroxylgroup. The particular removable protecting group employed is notcritical and preferred removable hydroxyl protecting groups includeconventional substituents such as benzyl, acetyl, chloroacetyl,benzylidine, t-butyl-diphenylsilyl and any other group that can beintroduced either enzymatically or chemically onto a hydroxylfunctionality and later selectively removed either by enzymatic orchemical methods in mild conditions compatible with the nature of theproduct. One such additional contemplated protecting group is aα-galactose which can be removed enzymatically with an α-galactosidase.

The term "pharmaceutically acceptable salts" includes thepharmaceutically acceptable addition salts of the compounds of Formula Iderived from a variety of organic and inorganic counter salts well knownin the art and include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetralkylammonium, and the like.

The term "aglycon" refer to the R substituent on the hexasaccharideglycosides of formula I and IV. In general, R is an aglycon having atleast 1 carbon atom. In a preferred embodiment, the aglycon moiety, R,is selected from the group consisting of --(A)--Z' wherein A representsa bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety ofthe formula --(CH₂ --CR₂ G)_(n) -- wherein n is an integer equal to 1 to5; R₂ is selected from the group consisting of hydrogen, methyl, orethyl; and G is selected from the group consisting of hydrogen, halogen,oxygen, sulphur, nitrogen, phenyl and phenyl substituted with 1 to 3substituents selected from the group consisting of amine, hydroxyl,halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbonatoms; and Z' is selected from the group consisting of hydrogen, methyl,phenyl, nitrophenyl, and, when G is not oxygen, sulphur or nitrogen andA is not a bond, then Z' is also selected from the group consisting of--OH, --SH, --NH₂, --NHR₃, --N(R₃)₂, --C(O)OH, --C(O)OR₃, --C(O)NH--NH₂,--C(O)NH₂, --C(O)NHR₃, --C(O)N(R₃)₂, and --OR₄ wherein each R₃ isindependently alkyl of from 1 to 4 carbon atoms and R₄ is an alkenylgroup of from 3 to 10 carbon atoms with the proviso that when A is abond, Z' is not hydrogen.

In those cases where the aglycon is one which permits linkage ofhexasaccharide glycoside I and/or IV to a carrier, then the aglycon ispreferably selected from the group consisting of --(A)--Z" wherein A isselected from the group consisting of an alkylene group of from 2 to 10carbon atoms and a moiety of the form --(CH₂ --CR₅ G)_(n) -- wherein nis an integer equal to 1 to 5; R₅ is selected from the group consistingof hydrogen, methyl, or ethyl; and G is selected from the groupconsisting of hydrogen, oxygen, sulphur, nitrogen, phenyl and phenylsubstituted with 1 to 3 substituents selected from the group consistingof amine, hydroxyl, halo, alkyl of from 1 to 4 carbon atoms and alkoxyof from 1 to 4 carbon atoms; and Z" is selected from the groupconsisting of hydrogen and, when G is not oxygen, sulphur or nitrogen,then Z" is also selected from the group consisting of --OH, --SH,--NH.sub. 2, --NHR₆, --C(O)OH, --C(O)OR₆, --C(O)NHNH₂, and --OR₇ whereineach R₆ is independently alkyl of from 1 to 4 carbon atoms and R₇ is analkenyl group of from 3 to 10 carbon atoms with the proviso that when Ais a bond, Z is not hydrogen. In such cases, the --(A)--Z" group definesa group capable of being linked to a carrier or is capable of beingderivatized to a group which is capable of being linked to a carrier.The choice of an appropriate carrier may be useful in enhancingimmunogenic properties.

Numerous aglycons are known in the art. For example, a linking armcomprising a para-nitrophenyl group (i.e., --OR=--OC₆ H₄ pNO₂) has beendisclosed by Ekberg et al.⁵⁰ At the appropriate time during synthesis,the nitro group is reduced to an amino group which can be protected asN-trifluoro-acetamido. Prior to coupling to a support, thetrifluoroacetamido group is removed thereby unmasking the amino group.

A linking arm containing sulfur is disclosed by Dahmen et al.⁵¹.Specifically, the linking arm is derived from a 2-bromoethyl groupwhich, in a substitution reaction with thionucleophiles, has been shownto lead to linking arms possessing a variety of terminal functionalgroups such as --OCH₂ CH₂ SCH₂ CO₂ CH₃ and --OCH₂ CH₂ SC₆ H₄ --pNH₂.

Rana et al.⁵² discloses a 6-trifluoroacetamidohexyl linking arm(--O--(CH₂)₆ --NHCOCF₃) in which the trifluoroacetamido protecting groupcan be removed unmasking the primary amino group used for coupling.

Other exemplification of known linking arms include the7-methoxycarbonyl-3,6,dioxaheptyl linking arm⁵³ (--OCH₂ --CH₂)₂ OCH₂ CO₂CH₃ ; the 2-(4-methoxycarbonylbutancarboxamido)ethyl⁵⁴ (--OCH₂ CH₂NHC(O)(CH₂)₄ CO₂ CH₃) the allyl linking arm⁵⁵ (OCH₂ CH═CH₂) which, byradical co-polymerization with an appropriate monomer,leads toco-polymers; other allyl linking arms⁵⁶ [--O(CH₂ CH₂ O)₂ CH₂ CH═CH₂ ].Additionally, allyl linking arms can be derivatized in the presence of2-aminoethanethiol⁵⁷ to provide for a linking arm --OCH₂ CH₂ CH₂ SCH₂CH₂ NH₂.

Additionally, as shown by Ratcliffe et al.⁵⁸, R group can be anadditional saccharide or an oligosaccharide containing a linking arm atthe reducing sugar terminus.

The carrier is generally a small molecular weight, non-immunogenic orantigenic carrier including the linking to a fluorescent label, aradioactive label, biotin, or a photolabile linking arm or a moiety tobe targeted.

In either case, the aglycon moiety is preferably a hydrophobic group andmore preferably a hydrophobic moiety selected from the group consistingof --(CH₂)₈ COOCH₃ and --(CH₂)₅ OCH₂ CH═CH₂. In particular, the use of ahydrophobic group and most especially, a --(CH₂)₈ COOCH₃ or --(CH₂)₅OCH₂ CH═CH₂ group may provide for some enhancement in the kinetics ofsialic acid transfer via a sialyltransferase.

As is apparent, hexasaccharide glycosides I and IV described above aredifferent from oligosaccharides and glycoconjugates because the aglyconmoiety (R) is not hydrogen, a protein, or a lipid capable of forming amicelle or other large aggregate structure.

B. SYNTHESIS AND METHODOLOGY B1. Preparation of Starting Materials

Tetrasaccharide glycosides II and V are readily prepared either bycomplete chemical synthesis or a chemical/enzymatic synthesis asdescribed below. Specifically, tetrasaccharide glycoside II and V can beprepared by chemically coupling the individual saccharide units. Suchcoupling can readily be prepared using a convergent synthesis, i.e.,appropriate saccharide units are linked together to form twodisaccharides which are then linked together to form a tetrasaccharide.Alternatively, the synthesis of tetrasaccharide glycosides can beconducted in a sequential synthesis starting with the saccharide unit atthe reducing sugar terminus and sequentially adding another saccharideunit until tetrasaccharide glycosides II and V are prepared.

In either case, the first step of the synthesis involves the addition ofthe aglycon moiety at the anomeric carbon atom of the reducing sugarunit. This is generally accomplished by using an appropriately protectedform of the reducing sugar and then selectively modifying this sugar atits anomeric center so as to introduce a leaving group comprisinghalides, trichloroacetimidate, thioglycoside, etc. The sugar donor isthen reacted under catalytic conditions (e.g., a soluble silver saltsuch as silver trifluoromethanesulfonate, a Lewis acid such as borontrifluoride etherate or trimethylsilyltrifluoromethanesulfonate, orthioglycoside promoters such as methyl trifluoromethanesulfonate ordimethyl(methylthio)-sulfonium trifluoromethanesulfonate) with anaglycon or an appropriate form of a carbohydrate acceptor which possessone free hydroxyl group at the position where the glycosidic linkage isto be established. See, for example, Paulsen²⁷, Schmidt²⁸, and Fugedi etal.²⁹, the disclosures of each of these references are incorporatedherein by reference in their entirety. A large variety of aglyconmoieties are known in the art and can be attached with the properconfiguration to the anomeric center of the reducing unit.

Appropriate use of compatible protecting groups, well known in the artof carbohydrate synthesis, will allow the further attachment of theother saccharide units. Each of the steps required to formtetrasaccharide glycosides II and V is well known in the art. Forexample, the synthesis of compound 1a (FIG. 1), i.e., the synthesis of aprotected form of a N-acetyllactosaminyl glycoside acceptor and itsglycosidation by an appropriate form of a N-acetyllactosaminyl donor arewell known in the art³⁰.

The synthesis of saccharide precursors having removable blockinggroup(s) is well known in the art and the removable blocking group canbe introduced at an appropriate stage during synthesis oftetrasaccharides II or V. For example, the selective opening of a4',6'--O--benzylidene of a glycoside and/or a thioglycoside of anappropriate form of a lactosamine disaccharide will provide thecorresponding 6'--O--benzyl derivative. The protected form of the6'--O--benzyl thioglycoside will be used as a donor in a glycosidationreaction leading to compound II, after deprotection. The appropriateform of the 6'-O-benzyl lactosaminyl glycoside will be used as acceptorin a glycosidation reaction leading to tetrasaccharide V afterdeprotection.

When the removable blocking group is sialic acid, then this group can bereadily introduced into tetrasaccharide VIII ##STR10## or intodisaccharide IX ##STR11## by use of a α(2-6)sialyltransferase asdescribed below.

B2. Enzymatic Sialylation

As noted above, deblocked (i.e., the removable blocking group X or X' isremoved) pentasaccharide glycoside III or blocked tetrasaccharideglycoside V ("oligosaccharide glycoside") is sialylated by contactingthe appropriate oligosaccharide glycoside with anα(2-3)sialyltransferase and a compatible CMP-derivative of a sialic acidor an analogue thereof under conditions wherein the sialic acid or thecompatible analogue thereof is transferred to the non-reducing sugarterminus of the oligosaccharide glycoside. Suitable conditions, known inthe art, include the addition of the appropriate sialyltransferase to amixture of the oligosaccharide glycoside and of the CMP-derivative ofthe sialic acid in an appropriate buffer such as 0.1 M sodium cacodylatein appropriate conditions of pH and temperature such as at a pH of 6.5to 7.5 and a temperature between 25° and 45° C., preferably 35°-40° C.for 12 hours to 4 days. The resulting sialylated oligosaccharideglycoside can be isolated and purified using conventional methodologycomprising HPLC, gel-, reverse phase-, ion exchange-, or adsorptionchromatography.

In this regard, when a compatible analogue of sialic acid is transferredto the oligosaccharide glycoside by the sialyltransferase, the analogueis sometimes referred to as an artificial donor and the oligosaccharideglycoside is sometimes referred to as an artificial acceptor.Sialylation methods employing an artificial donor and an artificialacceptor are described by Venot et al., U.S. patent application Ser. No.07/771,007 filed Oct. 2, 1992 which application is incorporated hereinby reference in its entirety. Similarly, sialylation methods employingan artificial donor and an artificial acceptor are described by Ippolitoet al.,²² which application is also incorporated herein by reference inits entirety.

The enzymatic transfer of compatible analogues of sialic acid requirethe prior synthesis (i.e., activation) of their nucleotide (CMP)derivatives. Activation of the analogues of sialic acid is usually doneby using the enzyme CMP-sialic acid synthase which is readily availableand the literature provides examples of the activation of variousanalogues of sialic acid.

The present invention is based, in part, on the discovery that, as shownin FIG. 1, sialylation of deblocked pentasaccharide glycoside III so asto form an α(2-3)sialyl residue at the non-reducing sugar terminus ofthis oligosaccharide glycoside is necessarily after removing theremovable blocking group because sialylation with anα(2-3)sialyltransferase requires the presence of a free hydroxyl groupat the 6-position of the terminal galactose residue on the deprotectedpentasaccharide glycoside III.

This invention is based, in part, on the further discovery that, asshown in FIGS. 2, the sialylation of the tetrasaccharide glycoside V soas to form an α(2-3)sialyl residue at the non-reducing sugar terminus ofthis oligosaccharide glycoside is necessarily before the fucosylationstep because sialylation with an α(2-3)sialyltransferase will notproceed if there is an α-fucose linked (1-3) to the neighboringN-acetylglucosamine.

B3. Enzymatic Fucosylation

As noted above, tetrasaccharide glycoside II or pentasaccharideglycoside derived by sialylating tetrasaccharide glycoside V ortrisaccharide 19 ("oligosaccharide glycoside") are fucosylated bycontacting the appropriate oligosaccharide glycoside with anα(1-3)fucosyltransferase and a compatible GDP-derivative of L-fucose oran analogue of L-fucose under conditions wherein the fucose istransferred onto the 3-hydroxy group of one of the GlcNAc moieties ofthe oligosaccharide glycoside. Suitable conditions, known in the art,include the addition of the α(1-3)fucosyltransferase to a mixture of theoligosaccharide glycoside and of the GDP-derivative of the L-fucose orcompatible analogue thereof in a appropriate buffer such as 0.1 M sodiumcacodylate in appropriate conditions of pH and temperature such as at apH of 6.5 to 7.5 and a temperature between 25° and 45° C., preferably35° to 40° C. for 12 hours to 4 days. The resulting fucosylatedoligosaccharide glycoside can be isolated and purified usingconventional methodology comprising HPLC, gel-, reverse phase-, ionexchange-, or adsorption chromatography.

As noted above, enzymatic fucosylation requires the prior synthesis ofGDP-fucose. Preferably, GDP-fucose is prepared in the methods describedby Jiang et al.⁵⁹

B4. Removal of the Removable Blocking Group

The synthesis of both hexasaccharides I and IV, as per FIGS. 1-2, bothrequire the removal of a removable blocking group. In general, theappropriate oligosaccharide glycoside is treated under conditionssufficient to effect removal of the blocking group. The specificconditions depend on the blocking group employed and are well known inthe art. For example, when a benzyl blocking group is employed, thisgroup is readily removed by hydrogenation techniques known in the art.Similarly, when the blocking group is sialic acid, it is removed in themanner depicted in the Examples set forth herein below.

Regarding FIGS. 1-2, FIG. 1 illustrates the synthesis of hexasaccharideglycoside I (compound 5a and 5b) and heptasaccharide glycoside (compound7a and 7b). Thus, the tetrasaccharide 1a was transformed into 2a byusing the βGal(1-4)βGlcNAc α(2-6) sialyltransferase from rat liver (FIG.1). It has been shown that a similar synthesis can be achieved on gramscale.¹⁶ Pentasaccharide 2a was then selectively fucosylated by theβGal(1-3)βGlcNAc α(13/4)fucosyltransferase [α(13/4)FT] from human milk⁴giving the hexasaccharides 3a,b Quantitative desialylation of 3a,b by asuitable immobilized sialidase (e.g., a sialidase from ClostridiumPerfringens) led to the fucosylated derivatives 4a,b, the ¹ H-n.m.r. ofwhich were in agreement with that of a synthetic material.¹⁷ The freeacid form 4b could be transformed into the methyl ester 4a by action ofdiazomethane in methanol. Desialylation of a glycolipid possessing ofthe same terminal hexasaccharide sequence has already beenmentioned.sup. 10. Finally, sialylation of 4a by the βGal(13/4)βGlcNAcα(2-3)sialyltransferase from rat liver provided the hexasaccharides5a,b.

The 8-methoxycarbonyloctyl glycoside of the starting tetrasaccharide 1aand trisaccharide 9 was used with the intention of taking advantage ofthe hydrophobic properties of the aglycone for separation purposes andwith the aim of possible coupling of the products to carriers.⁴ In fact,partial hydrolysis of the methyl ester could not really be avoided, andthis side reaction became important in some cases (e.g., when thetransferase such as milk fucosyltransferase was not highly purified). Asa result, compounds 3a, 4a, 5a, 6a and 7a were isolated as the methylester and/or the free acid forms (3b, 4b, 5b, 7b) of the aglycone whichwere identified by ¹ H-n.m.r. However, the free acid form of 4b canreadily be reconverted back to the methyl ester by treating the acid indry methanol with diazomethane.

Finally, although conversion of 1a into 2a appeared almost complete,some losses occurred during recovery of this derivative as it is notvery tightly retained on the hydrophobic C18 silica gel.

FIG. 1 also illustrates that heptasaccharide 7b was obtained bysequential sialylation of 1a by the βGal(13/4)βGlcNAcα(2-3)sialyltransferase, followed by difucosylation of the intermediate6a by the βGal(13/4)βGlcNAc α(13/4)fucosyltransferase from human milk.In the conditions used, only the difucosylated product was obtained.

FIG. 2 illustrates the synthesis of hexasaccharide IV (compound 12).

Alternative syntheses for hexasaccharide I are set forth below andgenerally involve a chemical/enzymatic approach. One approach is atotally enzymatic method which utilizes different glycosyltransferases.This procedure is set forth in FIG. 3. Specifically, in this approach,galactose is enzymatically transferred onto GlcNAc-OR to formβGal(1-4)βGlcNAc-OR (LacNAc-OR). Suitable enzymes include the GlcNAcβ1-4 galactosyltransferase which transfers galactose from uridine 5'(galactopyranosyl)-diphosphate (UDP-Gal) to the 4-position ofGlcNAcβ-OR, where R can be an aglycone or a saccharide. This transferaseis a commercial and versatile enzyme and accepts some modifications inthe sugar portion of the donor⁴⁷ and in the acceptor⁴⁸⁻⁴⁹.

N-acetylglucosamine is then transferred to the 3-position of theterminal β-galactose of LacNAc-OR(N-acetyllactosamine-OR--βGal(1-4)βGlcNAc-OR) to produce theβGlcNAc(1-3)βGal(1-4)βGlcNAc-OR trisaccharide structure (compound 19).Transferases which transfer N-acetylglucosamine from uridine5'-(N-acetylglucosamine)-diphosphate (UDP-GlcNAc) to the 3 position ofthe terminal β-galactose of a N-acetyllactosamine moiety) are present ina variety of sources such as human serum³⁶⁻⁴⁰, human urine⁴¹, Novikofftumor cell ascites fluid⁴²,43, mouse T-lymphoma cells⁴⁴, human milk⁴⁵and human colonic adenocarcinoma cells⁴⁶.

The acceptor specificity of the transferases obtained, particularly fromhuman serum³⁶,38 and from Novikoff tumor cell ascites fluid⁴³, has beenwell characterized using synthetic oligosaccharides. This enzymerequires a terminal βGal(1-4)βGlc(NAc)-OR unit, where R can be anaglycone or a saccharide moiety and Glc(NAc) can be either GlcNAc orGlc. The enzyme does not transfer to the structureβGal(1-4)[αFuc(1-3)]βGlcNAc (Lewis^(x)) in which the fucose is attachedto the penultimate GlcNAc³⁶,43. As a result, in this process, enzymaticformation of the βGlcNAc(1-3)βGal(1-4)βGlcNAc-OR should precedefucosylation. In addition, this enzyme, in combination with the GlcNAcβ(1-4) galactosyl-transferase could catalyze the synthesis of oligomersof N-acetyllactosamine⁴³.

Enzymatic transfer of fucose to the acceptor 19 by the milkGal(13/4)GlcNAc α(13/4)fucosyl-transferase specifically occurs on theinternal GlcNAc leading to compound 20. The backbone of thetetrasaccharide 20 is then extended by transfer of a galactose residueleading to compound 21 by the bovine GlcNAc β1-4 galactosyltransferase.Neu5Ac is then transferred to compound 21 (also shown in FIG. 1 ascompounds 4a,b) in the last step by the rat liver Gal(β13/4)GlcNAc α2-3sialyltransferase⁶⁰ providing hexasaccharide 22 (also shown in FIG. 1 ascompounds 5a,b).

As a result, the last three steps of this synthetic pathway, (1)fucosylation, (2) extension and (3) sialylation, differ from theproposed normal biosynthetic pathway which sequentially proceedsfollowing the sequence: (1) extension, (2) sialylation, and finally (3)fucosylation.

Alternatively, as shown in FIG. 4, compound 19 can be made in a totallysynthetic scheme starting from known precursors. Specifically, compound19 can be obtained by total chemical synthesis following knownprocedures by which a large variety of R group can be introduced. As aresult, R can be an aglycone or a saccharide moiety itself attached toan aglycone. A wide variety of glycosylation methods are available inorder to synthesize β-glycosides. The present synthesis is derived fromthe synthesis described by Alais et al.³⁰.

In addition, glycosidases can be used, instead of glycosyltransferases,for the synthesis of glycosides in appropriate conditions. The maincharacteristics of the use of both types of enzymes have been reviewedby Ichikawa et al.⁶². The β-galactosidases, N-acetylhexsaminidase orsialidases⁶³,64 could be used to synthesize some of the saccharides.

In these synthesis, the 2-N-phthalimido protecting group is used inorder to preferably lead to the β-glycosides during glycosylationreactions. Thus the blocked disaccharide glycosyl donor 11³⁰ is used ina glycosylation reaction of the desired alcohol ROH catalyzed bytrimethylsilyltrifluoromethanesulfonate to lead to the glycoside 12.Mild de--O--acetylation provided 13. Disaccharide 13 is reacted withacetone in the presence of an acid catalyst, such as p-toluene sulfonicacid at 60° C., leading to a mixture of the 3,4- and of the4,6-isopropylidene derivative 14 and 15 which are separated. The3,4-isopropylidene derivative 14 is totally acetylated in pyridine withacetic anhydride and the isopropylidene group hydrolyzed in a mixture ofacetic acid and water at 90° C. providing Glycosylation of diol 16 bythe donor 17 catalyzed by trimethylsilyltrifluoromethanesulfonatepreferably led to trisaccharide 18 which was deprotected usingconventional procedures leading to 19.

As set forth above, L-fucose is then transferred from GDP-fucose to thetrisaccharide acceptor 19 by using the Gal(β13/4)GlcNAc α13/4fucosyltransferase from human milk. Another appropriate transferase fromother source can also be used. As evidenced by ¹ H-n.m.r., only onefucosyl unit is introduced. Furthermore, the ¹ H-n.m.r. data, inparticular the position of the signal provided by H-5 of the αFuc ischaracteristic of the presence of the Gal(β1-4)[Fuc(α1-3)]GlcNAc unit⁶¹.The transformation is quantitative.

Galactose is then transferred from UDP-Gal to tetrasaccharide 20 by thecommercial bovine milk GlcNAc β1-4 galactosyltransferase. Thepentasaccharide obtained is identical to the same compound obtainedearlier by using a different route⁶⁵. The transformation isquantitative.

In a final step, Neu5Ac is transferred to pentasaccharide 21 by usingthe Gal(β13/4)GlcNAc α(2-3)sialyltransferase from rat liver²⁵. Anotherappropriate transferase from other sources can also be used. This stepis performed according to the earlier report and provides the sameproduct as described in Venot et al.⁶⁵.

As is apparent, more extended structures can be obtained from thepentasaccharide 21 as indicated in FIG. 5. For that purpose, GlcNAc canbe transferred to pentasaccharide 21 by the Gal(β1-4)GlcNAc β1-3N-acetylglucosaminyltransferase. Pentasaccharide 21 should be anacceptor for this transferase since the α-fucosyl residue is not linkedto the penultimate GlcNAc moiety. Further sequential transfer ofgalactose and of Neu5Ac by the appropriate glycosyltransferase will leadto octasaccharides 26.

C. Utility

Hexasaccharide glycosides I and IV are effective in suppressingmammalian cell-mediated immune responses. Without being limited to anytheory, it is believed that these compounds affect the cell mediatedimmune response in a number of ways. Specifically, these compounds caninhibit the ability of the immune response to become educated about aspecific antigen when the compound is administered simultaneously withthe first exposure of the immune system to the antigen. Also,hexasaccharide glycosides I and IV can inhibit the effector phase of acell-mediated immune response (e.g., the inflammatory component of a DTHresponse) when administered after second or later exposures of theimmune system to the same antigen. Additionally, hexasaccharideglycosides I and IV can induce tolerance to antigens when administeredat the time of second or later exposures of the immune system to theantigen.

The suppression of the inflammatory component of the immune response byhexasaccharide glycosides I and IV is believed to require the initiationof a secondary immune response (i.e., a response to a second exposure toantigen). Hexasaccharide glycoside I or IV is generally administered tothe patient at least about 0.5 hours after an inflammatory episode,preferably, at least about 1 hour after, and most preferably, at leastabout 5 hours after an inflammatory episode or exacerbation.

Hexasaccharide glycosides I and IV are effective in suppressingcell-mediated immune responses to an antigen (e.g., the inflammatorycomponent of a DTH response) when administered at a dosage range of fromabout 0.5 mg to about 50 mg/kg of body weight, and preferably from about0.5 to about 5 mg/kg of body weight. The specific dose employed isregulated by the particular cell-mediated immune response being treatedas well as by the judgment of the attending clinician depending uponfactors such as the severity of the adverse immune response, the age andgeneral condition of the patient, and the like. Hexasaccharideglycosides I or IV is generally administered parenterally, such asintranasally, intrapulmonarily, transdermally and intravenously,although other forms of administration are contemplated. Preferably, thesuppression of a cell-mediated immune response, e.g., the inflammatorycomponent of a DTH response, is reduced by at least about 10% as opposedto control measured 24 hours after administration of the challenge tothe mammal and 19 hours after administration of hexasaccharideglycosides I or IV.

In addition to providing suppression of the inflammatory component ofthe cell-mediated immune response to an antigen, administration of thehexasaccharide glycosides I or IV also imparts a tolerance to additionalchallenges from the same antigen. In this regard, re-challenge by thesame antigen weeks after administration of hexasaccharide glycosides Ior IV results in a significantly reduced immune response.

Administration of hexasaccharide glycosides I or IV simultaneously withfirst exposure to an antigen imparts suppression of a cell-mediatedimmune response to the antigen and tolerance to future challenges withthat antigen. In this regard, the term "reducing sensitization" meansthat the hexasaccharide glycosides I or IV, when administered to amammal in an effective amount along with a sufficient amount of antigento induce an immune response, reduces the ability of the immune systemof the mammal to become educated and thus sensitized to the antigenadministered at the same time as hexasaccharide glycosides I or IV. An"effective amount" of this compound is that amount which will reducesensitization (immunological education) of a mammal, including humans,to an antigen administered simultaneously as determined by a reductionin a cell-mediated response to the antigen such as DTH responses astested by the footpad challenge test. Preferably the reduction insensitization will be at least about 20% and more preferably at leastabout 30% or more. Generally, hexasaccharide glycosides I or IV areeffective in reducing sensitization when administered at a dosage rangeof from about 0.5 mg to about 50 mg/kg of body weight, and preferablyfrom about 0.5 mg to about 5 mg/kg of body weight. The specific doseemployed is regulated by the sensitization being treated as well as thejudgement of the attending clinician depending upon the age and generalcondition of the patient and the like. "Simultaneous" administration ofthe compound with the antigen with regard to inhibiting sensitizationmeans that the compound is administered once or continuously throughouta period of time within 3 hours of the administration of an antigen,more preferably the compound is administered within 1 hour of theantigen.

The methods of this invention are generally achieved by use of apharmaceutical composition suitable for use in the parenteraladministration of an effective amount of hexasaccharide glycosides I orIV. These compositions comprise a pharmaceutically inert carrier such aswater, buffered saline, etc. and an effective amount of hexasaccharideglycosides I or IV as to provide the above-noted dosage of theoligosaccharide glycoside when administered to a patient. It iscontemplated that suitable pharmaceutical compositions can additionallycontain optional components such as an adjuvant, a preservative, etc.

It is also contemplated that other suitable pharmaceutical compositionscan include oral compositions, transdermal compositions or bandagesetc., which are well known in the art.

Hexasaccharide glycosides I and IV containing an analogue of sialic acidare also useful in preparing artificial antigens which can cross-reactwith antigenic determinants having a similar oligosaccharide structureas in hexasaccharide glycosides I and IV but which contain a naturallyoccurring sialic acid. Methods for the preparation of artificialantigens and their uses are set forth in U.S. Ser. No. 07/771,007 filedconcurrently with this application as attorney docket number 005824-002and entitled "METHODS FOR THE ENZYMATIC SYNTHESIS OF ALPHA-SIALYLATEDOLIGOSACCHARIDE GLYCOSIDES" which application is incorporated herein byreference in its entirety.

The following examples are offered to illustrate the present inventionand are not to be construed in any manner as limiting it.

In these examples as well as in the application, all sugars disclosedare in their D form except for fucose which is in its L form.

In these examples, unless otherwise defined below, the abbreviationsemployed have their generally accepted meaning:

    ______________________________________                                        CMP-Neu5Ac =   cytidine-5'-monophospho-N-                                                    acetylneuraminic acid                                          DTH =          delayed-type hypersensitivity                                  Fuc T =        fucosyl transferase                                            Gal T =        galactosyl transferase                                         GDP-Fuc =      guanosine 5'-diphospho-L-fucose                                ST =           sialyl transferase                                             U =            Units                                                          UDP-Gal =      uridine-5'-diphospho-D-galactose                               ______________________________________                                         AG 1 × 8 (formate form) = ion exchange resin AG 1 × 8 (format     form) available from BioRad Laboratories, Richmond, CA                        Dowex 50W × 8 (H.sup.+  form) = ion exchange resin Dowex 50W .times     8 (H.sup.+  form) available from Dow Chemical, Midland, MI                    IRC50 resin (H.sup.+  form) = ion exchange resin IRC50 (H.sup.+  form)        available from Rohm & Haas, Philadelphia, PA                             

Commercially available components are listed by manufacturer and whereappropriate, the order number. Some of the recited manufacturers are asfollows:

Iatron=Iatron Laboratories, Tokyo, Japan

Merck=E. Merck AG, Darmstadt, Germany

Millipore=Millipore Corp., Bedford, Mass.

Waters=Waters Associates, Inc., Milford, Mass.

EXAMPLES

The following examples illustrate the preparation of Compounds 5a and 5bwhich preparation is illustrated in FIG. 1. The synthetic pathwayutilized the following general methods:

General Methods: All organic solvents used were re-distilled reagentgrade. Pre-coated silica gel plates (60-F254, E. Merck, Darmstadt) wererun in 65:35:5, 65:35:8 and/or 60:40:10 mixtures of CHCl₃, CH₃ OH, and0.2% CaCl₂ solution, and detection was by charring after spraying with a5% solution of sulphuric acid (H₂ SO₄) in ethanol. Sep-Pak C₁₈cartridges (Waters Associates, Milford, Mass.) were conditioned asindicated by the supplier. Iatrobeads (6RS-8060) were from IatronLaboratories, Tokyo, Japan and the AG 50W×8 ion exchange resin waspurchased from BioRad, Richmond, Calif. CMP-Neu5Ac was purchased fromSigma Chemical Company (St. Louis, Mo.) and GDP-fucose was obtained bychemical synthesis-⁵⁹ βGal(1-4)βGlcNAc(1-3)βGal-(1-4)βGlcNAc-OR wasobtained by following the procedures of Alais et al³⁰ with theappropriate substitution of the aglycon. Evaporation of organic solventswas done at 20°-25° C. using a rotory evaporator connected to a wateraspirator. ¹ H-n.m.r. spectra have been run on at 300 and 500 MHz usinginternal acetone (δ=2.225) as reference and samples were freeze driedtwice from D₂ O and dissolved in 99.99% D₂ O. The spectra of compoundsobtained as 8-methoxycarbonyloctyl glycosides all show a singlet atδ=3.686 (CO₂ CH₃) and a triplet at δ=2.387 (7.5 Hz, CH₂ CO₂). Thespectra of compounds obtained as the 8-carboxyoctyl glycosides differfrom the respective 8-methoxycarbonyloctyl glycosides by the absence ofthe singlet due to CO₂ CH₃ and the presence of a triplet at δ=2.314 (t,7.5 Hz) for CH₂ CO₂ H.

In examples 1 to 6 below, preparative sialylation was conducted asfollows:

The rat liver βGal(13/4)βGlcNAc α(2-3)sialyl-transferase (EC 2.4.99.5)was purified by affinity chromatography according to the procedure ofMazid, et al.¹⁹ but using a matrix obtained by covalently linking thehapten βGal(1-3)βGlcNAcO(CH₂)₈ CO₂ H⁶⁶ activated as in itsN-succinimidyl ester to epichlorohydrin activated Sepharose.⁶⁷

The βGal(1-4)-βGlcNAc α(2-6)sialyltransferase contained in theflow-through of the above affinity-column, was further chromatographedon CDP-hexanolamine Sepharose as reported.²⁰

The enzymatic sialylations were carried out at 37° C. in a plastic tubeusing a sodium cacodylate buffer (50 mM, pH 6.5) containing Triton CF-54(0.5%), BSA (1 mg/mL) and calf intestine alkaline phosphatase.²¹ Thefinal reaction mixtures were diluted with H₂ O and applied onto C₁₈Sep-Pak cartridges as reported.⁴ After washing with H₂ O, the productswere eluted with CH₃ OH and the solvents evaporated. The residue wasdissolved in a 65:35:5 mixture of CHCl₃, CH₃ OH and H₂ O and applied ona small column of Iatrobeads (0.200 to 0.500 g). After washing with thesame solvent mixture, the products were eluted with a 65:35:8 and/or65:40:10 mixtures of the same solvents. The appropriate fractions(t.l.c.) were pooled, the solvents evaporated in vacuo, the residue runthrough a small column of AG 50W×8 (Na⁺ form) in H₂ O and the productsrecovered after freeze drying in vacuo. In all cases, the8-methoxycarbonyloctyl glycosides were separated from the corresponding8-carboxyoctyl glycosides.

In examples 1 to 6 below, preparative fucosylation was conducted asfollows:

The βGlcNAc α(13/4)fucosyltranferase was purified from human milk, asreported.⁴ The enzymatic reactions were carried out at 37° C. in aplastic tube using a sodium cacodylate buffer (100 mM, pH 6.5), MnCl₂(10 mM), ATP (1.6 mM), NaN₃ (1.6 mM). The reaction products wereisolated and purified as indicated above.

Example 1 Preparation of 8-Methoxycarbonyloctyl(5-Acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-6)--O--β-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-glucopyranoside(2a)

Compound 1a (6.5 mg), CMP-Neu5Ac (17 mg), βGal(1-4)βGlcNAcα(2-6)sialyltransferase (50 mU) and alkaline phosphatase (15 U) wereincubated for 48 hours in 2.5 mL of the above buffer. Isolation andpurification provided 2a (3.0 mg).

Example 2 Preparation of 8-Methoxycarbonyloctyl(5-Acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-6)--O--β-D-galacto-pyranosyl-(1-4)--O--2-acetamido-2-deoxy-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O--]2-acetamido-2-deoxy-glucopyranoside(3a) and the 8-carboxyoctyl glycoside (3b)

Compound 2a (3.0 mg), GDP-fucose (5 mg), βGlcNAcα(13/4)fucosyltransferase (10 mU) were incubated for 68 hours in thebuffer (1.3 mL). Isolation and purification provided 3a (1.2 mg) and 3b(0.5 mg).

Example 3 Preparation of 8-Methoxycarbonyloctylβ-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O--]2-acetamido-2-deoxy-β-D-glucopyranoside(4a) and the 8-carboxyoctyl glycoside (4b)

Compounds 3a and 3b (1.7 mg) were incubated with Clostridium Perfringensneuraminidase immobilized on agarose (Sigma Chemical Company, 1 U) in abuffer of sodium cacodylate (50 mM, pH 5.2, 2 mL) at 37° C. After 24hours the mixture was diluted with water (10 mL) and filtered throughAmicon PM-10 membrane. The flow-through and washings were lyophilizedand the residue dissolved in water (3 mL) and applied to two C₁₈cartridge. Each cartridge was washed with water (10 mL) prior to elutionwith methanol (20 mL). After evaporation of the solvent, the residue waschromatographed on Iatrobeads (210 mg) as indicated above giving (4a,0.8 mg) and 4b (0.7 mg). 4b was dissolved in dry methanol and treatedwith diazomethane until t.l.c. indicated the complete conversion into4a.

Example 4 Preparation of 8-Methoxycarbonyloctyl(5-Acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)--O--β-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O--]2-acetamido-2-deoxy-.beta.-D-glucopyranoside(5a) and the 8-carboxyoctyl glycoside (5b)

Compound 4a (1.5 mg), CMP-Neu5Ac (8 mg), βGal(13/4)βGlcNAcα(2-3)sialyltransferase (17 mU), alkaline phosphatase (5 U), wereincubated for 40 hours in the sialylation buffer (1.5 mL). Isolation andpurification provided 5a (0.7 mg) and 5b (0.55

Example 5 Preparation of 8-Methoxycarbonyloctyl(5-Acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)--O--β-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-.beta.-D-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--2-acetamido-2-deoxy-glucopyranoside(6)

Compound 1a (5 mg), CMP-Neu5Ac (15 mg), βGal(13/4)βGlcNAcα(2-3)sialyltransferase (46 mU), and alkaline phosphatase (15 U) wereincubated in the sialylation buffer (2,5 mL) for 48 hours. Isolation andpurification of the product gave 6a (2.5 mg).

Example 6 Preparation of 8-Methoxycarbonyloctyl(5-Acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)--O--β-D-galactopyranosyl-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O--]2-acetamido-2-deoxy-glucopyranosyl-(1-3)--O--β-D-galactopyranosyl-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O--]2-acetamido-2-deoxy-glucopyranoside(7a)

Compound 6a (2.5 mg), GDP-fucose (8 mg) and the βGlcNAcα(1→3/4)fucosyltransferase (19 mU) were incubated in the enzymaticbuffer (2.0 mL) for 48 h. Isolation and purification of the product give7b (1.7 mg).

¹ H-NMR data for the compounds prepared in Examples 1 to 6 above are setforth in the following Table I:

                                      TABLE I                                     __________________________________________________________________________    .sup.1 H-n.m.r. Structural Parameters                                         Sugar Unit                                                                            Hydrogen                                                                            1a    2a    3a    4a  5a    6a      7b                          __________________________________________________________________________    βGlcNAc                                                                          1 (d) 4.516 (8.2)                                                                         4.516 (7.5)                                                                         4.522 (8.0)                                                                         4.525                                                                             4.526 (8.0)                                                                         4.516 (7.5)                                                                           4.527 (7.8)                                                 (7.8)                                         βGal                                                                             1 (d) 4.457* (7.8)                                                                        4.455* (7.8)                                                                        4.439 (7.8)                                                                         4.436                                                                             4.435 (8.0)                                                                         4.457 (7.7)                                                                           4.434 (7.7)                         4 (d) 4.157 (3.0)                                                                         4.417 (3.2)                                                                         4.095 (3.0)                                                                         (7.7)                                                                             4.098 (3.0)                                                                         4.156 (3.2)                                                                           4.092 (3.2)                                                 4.098                                                                         (3.2)                                         βGlcNAc                                                                          1 (d) 4.698 (8.2)                                                                         4.728 (7.7)                                                                         4.722 (7.7)                                                                         4.703                                                                             4.692 (8.0)                                                                         4.696 (8.0)                                                                           4.690 (8.3)                                                 (7.7)                                         βGal                                                                             1 (d) 4.479* (7.8)                                                                        4.462* (7.8)                                                                        4.455 (8.0)                                                                         4.480                                                                             4.457 (7.7)                                                                         4.555 (9.0)                                                                           4.527 (7.8)                         3 (dd)                  (7.7)                                                                             4.114 (3.0,                                                                         4.114 (3.0, 9.8)                                                                      4.082 (3.0, 10.0)                                               10.0)                                     αFuc                                                                            1 (d)             5.094 (3.7)                                                                         5.096                                                                             5.094 (3.8)   5.127, 5.093                        5 (q)             4.814 (6.5)                                                                         (3.8)                                                                             4.814 (6.5)   (3.8)                               6 (d)             1.150 4.814                                                                             1.150         4.822, 4.818                                                (6.5)             (6.5)                                                       1.152             1.170, 1.144                αNeu5Ac(2-3)                                                                    3.sub.ax (dd)               2.756 (4.5,                                                                         2.758 (4.5,                                                                           2.762 (4.5, 12.7)                   3.sub.eq (t)                13.0) 12.5)   1.792 (12.0)                                                    1.796 (12.0)                                                                        1.796 (12.2)                        αNeu5Ac(2-6)                                                                    3.sub.ax (dd)                                                                             2.670 (4.5,                                                                         2.666 (4.5,                                                 3.sub.eq (t)                                                                              12.5) 12.5)                                                                   1.720 (12.0)                                                                        1.718 (12.0)                                        NAc     (s)   2.028 2.027 (two)                                                                         2.021, 2.026                                                                        2.027                                                                             2.024 2.030   2.012, 2.018                              2.033 2.055 2.043 2.032                                                                             (three)                                                                             (three) 2.028                       CH.sub.2 CO.sub.3                                                                     (t)   2.388 (7.5)                                                                         2.387 (7.5)                                                                         2.387 (7.5)                                                                         2.386                                                                             2.386 (7.5)                                                                         2.387 (7.5)                                                                           2.314 (7.5)                                                 (7.5)                                         CO.sub.2 R    CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                          CH.sub.3                                                                            CH.sub.3                                                                              H                                         3.685 3.686 3.686 3.688                                                                             3.686 3.684                               __________________________________________________________________________     *interchangeable                                                         

KINETIC DATA

Experiments were conducted to determine the relative rates of transferof fucosylation onto the 3-hydroxy of the GlcNAc in disaccharideglycosides βGal(1-4)βGlcNAc-OR having different substituents at the6-position of the galactose. Fucosyltransferase assays were conductedwith α(13/4)fucosyltransferase in a manner similar to that described inthe art⁴ and gave the following results:

    ______________________________________                                        Substituent at                                                                the 6-position              Relative Rate                                     of galactose                                                                             R                of Transfer                                       ______________________________________                                        --OH       --(CH.sub.2).sub.8 CO.sub.2 CH.sub.3                                                           100                                               H          --(CH.sub.2).sub.2 O(CH.sub.2).sub.2 CO.sub.2 CH.sub.3                                          9                                                ______________________________________                                    

The remaining low relative rate of transfer obtained on the 6'-deoxyderivative may be due to a small amount of βGal α(1-2)fucosyltransferasewhich was present in the preparation of the α(13/4)fucosyltransferase.

The above results indicate that the presence of a hydroxyl group at the6-position of galactose is necessary for efficient fucosylation of3-hydroxy of the GlcNAc in disaccharide glycosides βGal(1-4)βGlcNAc-ORusing α(13/4)fucosyltransferase.

The following Examples 7 to 8 illustrate alternative methods forpreparing for compounds of Formula I.

In these examples, during the chemical synthesis, unless otherwisespecially indicated, the work up generally included extraction withdichloromethane followed by the normal sequential washings of theorganic phase with water, a dilute solution of sodium carbonate andwater. The organic solvent were then dried over magnesium sulfate, thesolid filtered and the solvent evaporated in vacuo as indicated.

Evaporation of organic solvents was done at 20°-25° C. using a rotoryevaporator connected to a water aspirator. ¹ H-n.m.r. spectra were runat 300 MHz using internal acetone (δ=2.225) as reference and sampleswere freeze dried twice from D₂ O and dissolved in 99.99% D₂ O. Thespectra of compounds obtained as 8-methoxycarbonyloctyl all show asinglet at δ=3.686 (CO₂ CH₃) and a triplet at δ=2.387 (7.5 Hz, CH₂ CO₂).The spectra of compounds obtained as the 8-carboxyoctyl glycosidesdiffer from the respective 8-methoxycarbonyloctyl glycosides by theabsence of the singlet due to CO₂ CH₃ and the presence of a triplet atδ=2.314 (t, 7.5 Hz) for CH₂ CO₂ H.

Preparative Enzymatic Sialylation

The rat liver βGal(13/4)βGlcNAc α(2-3)sialyltransferase (EC 2.4.99.5)was purified by affinity chromatography according to the procedure ofMazid, et al.¹⁹ but using a matrix obtained by covalently linking thehapten βGal(1-3)βGlcNAcO(CH₂)αCO₂ H⁶⁶ activated as in its N-succinimidylester to epichlorohydrin activated Sepharose.⁶⁷ The enzymaticsialylations were carried out at 37° C. in a plastic tube using a sodiumcacodylate buffer (50 mM, pH 6.5) containing Triton CF-54 (0.5%), BSA (1mg/mL) and calf intestine alkaline phosphatase.⁶⁹ The final reactionmixtures were diluted with H₂ O and applied onto C₁₈ Sep-Pak cartridgesas reported.⁴ After washing with H₂ O, the products were eluted with CH₃OH and the solvents evaporated. The residue was dissolved in a 65:35:5mixture of CHCl₃, CH₃ OH and H₂ O and applied on a small column ofIatrobeads (0.200 to 0.500 g). After washing with the same solventmixture, the products were eluted with a 65:35:8 and/or 65:40:10mixtures of the same solvents. The appropriate fractions (t.l.c.) werepooled, the solvents evaporated in vacuo, the residue run through asmall column of AG 50W×8 (Na⁺ form) in H₂ O and the products recoveredafter freeze drying in vacuo. In all cases, the 8-methoxycarbonyloctylglycosides were separated from the corresponding 8-carboxyoctylglycosides.

Preparative Enzymatic Fucosylation Enzymatic Conditions

The βGlcNAc α(13/4)fucosyltransferase (EC 2.4.1.65) was purified fromhuman milk, as reported.⁴ The enzymatic reactions were carried out at37° C. in a plastic tube using a sodium cacodylate buffer (100 mM, pH6.5), MnCl₂ (10 mM), ATP (1.6 mM), NaN₃ (1.6 mM). The reaction productswere isolated and purified as indicated above.

Synthesis of GDP-Fucose A. Preparation of Bis(tetra-n-butylammonium)hydrogen phosphate

Tetra-n-butylammonium hydroxide (40% aq. w/w, about 150 g) was addeddropwise to a solution of phosphoric acid (85% aq, w/w, 18 g, 0.155mmol) in water (150 mL) until the pH reached 7. Water was thenevaporated in vacuo to give a syrup which was co-evaporated with dryacetonitrile (2×400 mL) followed by dry toluene (2×400 mL). Theresulting white solid (75 g) was dried in vacuo and stored overphosphorus pentoxide under vacuum until used.

B. Preparation of β-L-Fucopyranosyl-1-Phosphate

A solution of bis(tetra-n-butylammonium) hydrogen phosphate (58 g, 127.8mmol) in dry acetonitrile (300 mL) was stirred at room temperature undernitrogen in the presence of molecular sieves (4Å, 20 g) for about onehour. A solution of tri-O-acetyl fucosyl-1-bromide (freshly preparedfrom 31 g, 93 mmol of L-fucose tetraacetate in the manner of Nunez etal.⁷²) in dry toluene (100 mL) was added dropwise in about 0.5 hour tothe above solution, cooled at 0° C. After one more hour at 0° C., themixture was brought to room temperature and stirred for 3 hour. Tlc (1:1toluene:ethyl acetate) indicated a main spot on the base line andseveral faster moving smaller spots.

The mixture was filtered over a pad of Celite (which was further washedwith acetonitrile) and the solvents evaporated in vacuo to give a redsyrup. This material was dissolved in water (400 mL) and extracted withethyl acetate (250 mL, twice). The aqueous layer was then evaporated invacuo leaving a yellowish syrup to which a solution of ammoniumhydroxide (25% aq., 200 mL) was added. The mixture was stirred at roomtemperature for 3 hours after which tlc (65:35:8chloroform:methanol:water) indicated a baseline spot. The solvent wasevaporated in vacuo to give a yellowish syrup which was diluted withwater (400 mL). The pH of this solution was checked and brought to 7, ifnecessary, by addition of a small amount of hydrochloric acid. Thesolution was slowly absorbed onto a column of ion exchange resin Dowex2×8 [200-400 mesh, 5×45 cm, bicarbonate form which had been prepared bysequential washing of the resin with methanol (800 mL), water (1200 mL),ammonium bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water (1000mL) was then run through the column followed by a solution of ammoniumbicarbonate (0.5 M, 2.3 mL/minute, overnight). The eluate was collectedin fractions (15 mL) and the product detected by charting after spottingon a tlc plate. Fractions 20 to 57 were pooled and evaporated in vacuoleaving a white solid which was further co-evaporated with water (3×300mL) and freeze drying of the last 50 mL and then drying of the residuewith a vacuum pump to give β-L-fucopyransyl-1-phosphate (9.5 g, 40%) asa 12:1 mixture of β and α anomers containing some ammonium acetateidentified by a singlet at δ=1.940 in the ¹ H-n.m.r. spectrum. Thisproduct was slowly run through a column of Dowex 5×8 resin (100-200mesh, triethylammonium form) and eluted with water to provide the bistriethylammonium salt of β-L-fucopyransyl-1-phosphate as a sticky gumafter freeze drying of the eluate. ¹ H-n.m.r. δ:4.840 (dd, J₁,2 =J₁,p =7.5 Hz, H-1), 3.82 (q, 1H, J₅,6 6.5 Hz, H-5), 3.750 (dd, 1H, J₃,4 3.5,J₄,5 1.0 Hz, H-4), 3.679 (dd, 1H, J₂,3 10.0 Hz, H-3), 3.520 (dd, 1H,H-2), 1.940 (s, acetate), 1.26 (d, H-6). Integral of the signals at 3.20(q, J 7.4 Hz, NCH₂) and 1.280 and 1.260 (NCH₂ CH₃ and H-6) indicatesthat the product is the bis-triethylammonium salt which may loose sometriethylamine upon extensive drying. ¹³ C-n.m.r. δ:98.3 (d, J_(C),1P 3.4Hz, C-1), 72.8 (d, J_(C),2P 7.5 Hz, C-2), 16.4 (C-6); ³¹ P-nmrδ:+2.6(s).

β-L-fucopyransyl-1-phosphate appears to slowly degrade upon prolongedstorage (1+ days) in water at 22° C. and, accordingly, the materialshould not be left, handled or stored as an aqueous solution at 22° C.or higher temperatures. In the present case, this material was kept at-18° C. and dried in vacuo over phosphorus pentoxide prior to being usedin the next step.

C. Preparation of Guanosine 5'-(β-1-Fucopyranosyl)-Diphosphate

Guanosine 5'-(β-1-fucopyranosyl)-diphosphate was prepared fromβ-L-fucopyranosyl-1-phosphate using two different art recognizedprocedures as set forth below:

PROCEDURE #1

β-L-fucopyranosyl-1-phosphate and guanosine 5'-mono-phosphomorpholidate(4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt, available fromSigma, St. Louis, Mo., "GMP-morpholidate") were reacted as described ina recent modification⁷⁴,75 of Nunez's original procedure⁷ ₂.Accordingly, tri-n-octylamine (0.800 g, available from Aldrich ChemicalCompany, Milwaukee, Wis.) was added to a mixture ofβ-L-fucopyranosyl-1-phosphate (triethylammonium salt, 1.00 g, about 2.20mmol) in dry pyridine (10 mL) under nitrogen the solvent removed invacuo. The process was repeated three times with care to allow only dryair to enter the flask. GMP morpholidate (2.4 g, about 3.30 mmol) wasdissolved in a 1:1 mixture of dry dimethylformamide and pyridine (10mL). The solvents were evaporated in vacuo and the procedure repeatedthree times as above. The residue was dissolved in the same mixture ofsolvents (20 mL) and the solution added to the reaction flaskaccompanied by crushed molecular sieves (2 g, 4Å). The mixture wasstirred at room temperature under nitrogen. Tlc (3:5:2 25% aq. ammoniumhydroxide, isopropanol and water) showed spots corresponding to thestarting GMP-morpholidate (Rf˜0.8, U.V.), guanosine5'-(β-1-fucopyranosyl)-diphosphate (Rf˜0.5, U.V. and charring), followedby the tailing spot of the starting fucose-1-phosphate (Rf˜0.44,charring). Additional U.V. active minor spots were also present. Afterstirring for 4 days at room temperature, the yellowish mixture wasco-evaporated in vacuo with toluene and the yellowish residue furtherdried overnight at the vacuum pump leaving a thick residue (2.43 g).Water (10 mL) was then added into the flask to give a yellow cloudysolution which was added on top of a column of AG 50W-X12 (from Biorad)resin (100-200 mesh, 25×1.5 cm, Na⁺ form). The product eluted with waterafter the void volume. The fractions which were active, both by U.V. andcharring after spotting on a tlc plate, were recovered and the solutionfreeze-dried overnight in vacuo providing a crude material (1.96 g).

This residue was dissolved in water (10 mL overall) and slowly absorbedonto a column of hydrophobic C₁₈ silica gel (Waters, 2.5×30 cm) whichhad been conditioned by washing with water, methanol and water (250 mLeach). Water was then run through the column (0.4 mL/min) and the eluatecollected in fractions (0.8 mL) which were checked by tlc (3:5:2 25% aq.ammonium hydroxide, isopropanol and water).β-L-fucopyranosyl-1-phosphate, (Rf˜0.54, charting) was eluted infractions 29 to 45. A product showing a strongly U.V. active spot(Rf˜0.51) eluted mainly in fractions 46 to 65. Other minor U.V. activespots of higher or lower Rf were observed. Fractions 59 to 86, whichcontained guanosine 5'-(β-1-fucopyranosyl)-diphosphate (Rf˜0.62), alsoshowed a narrow U.V. active spot (Rf˜0.57). Fractions 59 to 86 werepooled and freeze-dried overnight providing 0.353 g of material enrichedin guanosine 5'-(β-1 -fucopyranosyl)-diphosphate. ¹ H-n.m.r. indicatedthat this material was contaminated by a small amount of impuritiesgiving signals at δ=4.12 and δ=5.05.

Fractions 29 to 45 and 47 to 57 were separately pooled and freeze-driedproviding recovered β-L-fuco-pyranosyl-1-phosphate (0.264 g and 0.223 g,respectively, in which the second fraction contains some impurities).Occasionally, pooling of appropriate fractions provided some amount ofguanosine 5'-(β-1-fucopyranosyl)-diphosphate in good purity (¹H-n.m.r.). Generally, all the material enriched in guanosine5'-(β-1-fuco-pyranosyl)diphosphate was dissolved in a minimum amount ofwater and run on the same column which had been regenerated by washingwith large amounts of methanol followed by water. The fractionscontaining the purified guanosine 5'-(β-1-fucopyranosyl)-diphosphate(tlc) were pooled and freezed dried in vacuo leaving a white fluffymaterial (187 mg, 16%). ¹ H-n.m.r. was identical to the previouslyreported data⁷³.

PROCEDURE #2

β-L-fucopyranosyl-1-phosphate and guanosine 5'-monophosphomorpholidate(4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt--"GMP-morpholidate") were reacted in dry pyridine as indicated in theoriginal procedure²². Accordingly, the β-L-fucopyranosyl-1-phosphate(triethylammonium salt, 0.528 g, about 1.18 mmol) was dissolved in drypyridine (20 mL) and the solvent removed in vacuo. The process wasrepeated three times with care to allow only dry air to enter the flask.GMP-morpholidate (1.2 g, 1.65 mmol) and pyridine (20 mL) were added intothe reaction flask, the solvent evaporated in vacuo and the processrepeated three times as above. Pyridine (20 mL) was added to the finalresidue and the heterogeneous mixture was stirred for 3 to 4 days atroom temperature under nitrogen. An insoluble mass was formed which hadto be occasionally broken down by sonication.

The reaction was followed by tlc and worked up as indicated in the firstprocedure to provide the GDP-fucose (120 mg, 16%).

Preparative Enzymatic Galactosylation

The bovine milk βGlcNAc 5(1-4) galactosyltransferase (EC 2.4.1.22,specific activity 6.5 units/mg of protein) and UDP-Gal were obtainedfrom Sigma. The enzymatic reactions were carried out at 37° C. in aplastic tube using a sodium cacodylate buffer (100 mM, pH 7.5)containing 20 mM manganese dichloride. The reaction products werepurified as indicated above in the case of the preparative sialylation.

In some cases, depending upon the enzymatic preparation, it may happenthat the terminal methyl ester of the aglycone is hydrolyzed. As aresult, the final products may possibly be isolated as saccharidespossessing the aglycone terminated by a methyl ester or a free acidgroup. These two saccharides are separated during the step of thechromatography on Iatrobeads as indicated above. The two forms of theaglycone of the saccharide are identified by ¹ H-n.m.r.

Example 7 Synthesis of 8-Methoxycarbonyloctyl(2,3,4,6-tetra--O--acetyl-β-D-galactopyranosyl)-(1-4)--O--3,6-di--O--acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(Compound 19) A. Synthesis of Compound 12

A solution of trimethylsilyltrifluoromethanesulfonate (0.504 mL, 2.6mmol) in dichloromethane (4 mL) was added to the mixture of thedisaccharide donor 11³⁰ (2.0 g, 2.6 mmol), drierite (4.0 g, crushed) and8-methoxycarbonyl-octanol (1.9 g, 10.0 mmol) in dichloromethane (30 mL)at 4° C. After stirring for 0.5 h at 4° C., the mixture was slowlywarmed up to room temperature for 1 h. After cooling to 4° C., a secondportion of the catalyst (0.250 mL, 1.3 mmol) in dichloromethane (2 mL)was added. After slowly warming up and stirring at room temperature for1 h, the reaction was stopped by addition of triethylamine. Afterfiltration, the crude product recovered after the usual work up wasdried in vacuo, and acetylated in a 2:1 mixture of pyridine and aceticanhydride. After addition of methanol, the mixture was worked up asusual, and the solvents co-evaporated with an excess of toluene. Theresidue was chromatographed on silica gel using a 2:1 mixture of tolueneand ethyl acetate providing compound 12 (1.40 g, 60%). ¹ H-n.m.r.(CDCl₃): δ7.90-7.70 (m, 4H, aromatics), 5.75 (dd, 1H, J₂,3 10.5 J₃,49.5Hz, H-3), 5.34(m, 2H, incl. H-1 and H-4'), 5.15(dd, 1H, J_(1'),2'8.0, J_(2'),3' 10.5 Hz, H-2'), 4.97(dd, 1H, H-3'), 3.67(s, 3H, CO₂ CH₃),2.25(t, 2H, J 7.5 Hz, CH₂ CO₂), 2.19-1.94(6s, 18H, 60Ac), 1.45 (m, 4H)and 1.08 (m, 8H): methylenes.

Synthesis of Compound 5--8-Methoxycarbonyloctyl(2,6-di--O--acetyl-3,4--O--isopropylidene-β-D-galactopyranosyl)-(1-4)--O--(3,6-di--O--acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(15)

A 1M solution of sodium methoxide in methanol (0.200 mL) was added to asolution of compound 12 (1.40 g, 1.65 mmol) in methanol (40 mL) cooledat 4° C. After 1.5 h at 4° C., the solution was deionized using IRC-50resin (H⁺ form). The resin was filtered, the solvent evaporated and theproduct dried in vacuo (1.0 g, 94%).

A solution of the above material (0.776 g, 1.2 mmol) and paratoluenesulfonic acid monohydrate (60 mg) in dry acetone (60 mL) was refluxedfor 3 h. After neutralization with triethylamine, the solvent wasevaporated and the residue chromatographed on silica gel using a 100:1mixture of ethyl acetate and methanol providing compound 14 (0.575 g,70%); ¹ H-n.m.r. (CD₃ OD, DOH at 4.80): 7.80-7.60 (m, 4H, aromatics),5.10 (d, 1H, J₁,2 8.0 Hz, H-1), 4.38 (m, 2H, H-1 and H-3), 3.70(s, 3H,CO₂ CH₃), 2.31(t, J 7.5 Hz, CH₂ CO₂), 1.65-1.00[m, incl. 1.57 and 1.45(2s, C(CH₃)₂ ]. Further elution provided the 4,6-isopropylidenederivative (0.200 g, 24%).

Compound 14 (0.515 g, 0.84 mmol) was acetylated in a 2:1 mixture ofpyridine and acetic anhydride for 24 h at 22°. After addition ofmethanol and the usual work up, the solvents were co-evaporated with anexcess of toluene and the residue chromatographed on silica gel using a100:3 mixture of chloroform and methanol providing compound 15 (0.646 g,90%); [α]_(D) +13.8° (c, 1 chloroform); ¹ H-n.m.r. (CDCl₃); δ7.90-7.70(m, 4H, aromatics), 5.74 (J₁,2 8.5,, J₂,3 10.5 Hz, H-3), 5.34 (d, 1HJ₁,2 8.5 Hz, H-1), 4.88 (dd, 1H, J_(1'),2' ˜J_(2'),3' 6.5 Hz, H-2'),3.67(s, 3H, CO₂ CH₃), 2.23(t, J 7.5 Hz, CH₂ CO₂), 2.14, 2.13, 2.10,1.91(4s, 12H, 4 OAc), 1.30-1.54 [m, incl. 1.53 and 1.32 (2s, C(CH₃)₂ ].

C. Synthesis of Compound 16--8-Methoxycarbonyloctyl(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1-4)--O--3,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside

Compound 15 (0.575 g, 0.68 mmol) in 90% acetic acid (12 mL) was heatedat 80° for 2 h. After dilution with dichloromethane, the solvent waswashed with water, a solution of sodium bicarbonate and water. Afterdrying over magnesium sulfate, the solvents were evaporated in vacuo,and the residue chromatographed on silica gel providing compound 16(0.452 g, 82%); [α]_(D) +12.1 (c, 1.03 chloroform).

D. Synthesis of Compound 8--8-Methoxycarbonyloctyl(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1-3)--O--(2,6-di--O--acetyl-β-D-galactopyranosyl)-(1-4)--O--3,6-di--O--acetyl-2-deoxy-2-phthlamido-β-D-glucopyranoside(18)

Trimethylsilyltrifluoromethanesulfonate (0.036 mL, 0.060 mmol) inmethylene chloride (0.5 mL) was added to a solution of compound 16(0.100 g, 0.123 mmol) in methylene chloride (5 mL). A solution of theimidate 17 (0.102 g, 0.185 mmol) in methylene chloride (4 mL) was slowlyadded to the above solution cooled at -70°. The mixture was furtherstirred at that temperature for 0.5 h. An additional portion of thecatalyst (0.018 mL, 0.030 mmol) in methylene chloride (0.5 mL) wasfurther added. After 0.5 h at -70°, the reaction was stopped by additionof triethylamine, and the mixture worked up as usual. The recoveredresidue was chromatographed on silica gel using a 100:2 mixture ofchloroform and methanol providing compound 18 (0.120 g, 80%); ¹ H-n.m.r.(CDCl₃): δ7.95-7.60 (m, 8H, aromatics), 5.74 (dd, 1H, J_(2"),3" 10.5J_(3"),4" 9.0 Hz, H-3"), 5.61(dd, 1H, J₂,3 10.5, J₃,4 8.5 Hz, H-3),5.48(d, 1H, J_(1"),2" 8.5 Hz, H-1"), 5.27 (d, 1H, J₁,2 8.5 HZ, H-1),5.14 (dd, 1H, J_(4"),5" 10.0 Hz, H-4"), 4.90(dd, 1H, J_(1'),2' 8.0J_(3'),4' 10.0 Hz, H-2'), 3.68(s, CO₂ CH₃), 0.22(t, J 7.5 Hz, CH₂ CO₂),2.12(two), 2.10, 2.04, 1.86, 1.85, 1,56(6s, 21H, 7 OAc), 1.40 (m, 4H),and 1.20 (m, 8H): methylenes.

E. Synthesis of Compound 19--8-Methoxycarbonyloctyl(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)--O--(β-D-galactopyranosyl)-(1-4)--O--2-acetamido-2-deoxy-β-D-glucopyranoside

Hydrazine acetate (1.27 g, 13.8 mmol) was added to compound 18 (0.120 g,0.098 mmol) in anhydrous ethanol (15 mL). The mixture was refluxed for18 h. The solvents were then co-evaporated with an excess of toluene.After drying in vacuo, the residue was acetylated in a 2:1 mixture ofpyridine and acetic anhydride for 48 h. After quenching the excess ofacetic anhydride with some methanol, the reaction mixture was worked upas usual. The recovered solvents were evaporated in vacuo and theresidue co-evaporated with an excess of toluene. The residue waschromatographed on silica gel using a 100:9 mixture of chloroform andmethanol as eluant provided the peracetylated trisaccharideintermediate. This material was de--O--acetylated in anhydrous methanol(5 mL) in the presence of 0.2 M sodium methoxide in methanol (0.200 mL).After overnight at 22° C., de-ionization with Dowex 50×8 and filtration,the solvent was evaporated in vacuo. The recovered product waschromatographed on BioGel P-2 and eluted with a 1:1 mixture of water andethanol which provided the pure trisaccharide 19 (0.044 g, 60%); [α]_(D)-4.8° (c, 0.48, water); ¹ H-n.m.r. (D₂ O): data provided in Table II.

Example 8 Synthesis of 8-Methoxycarbonyloctyl(5-acetamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)--O--(β-D-galactopyranosyl)-(1-4)--O--(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)--O--(β-D-galactopyranosyl)-(1-4)--O--[β-L-fucopyranosyl-(1-3)--O]-2-acetamido-2-deoxy-β-D-glucopyranoside(22) (Compound) 22--the CD-65/VIM-2 Saccharide) A. Synthesis of Compound20--8-Methoxycarbonyloctyl(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)--O--(β-D-galactopyranosyl)-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O]-2-acetamido-2-deoxy-β-D-glucopyranoside

Compound 19 (15 mg), GDP-fucose (33 mg) and the βGlcNAcα(13/4)fucosyltransferase (56 mU) were incubated for 72 hours in thebuffer (4 mL) as indicated above. Isolation and purification providedthe compound 20 (14.0 mg). ¹ H-n.m.r. data is included in Table II.

B. Synthesis of Compound 21--8-Methoxycarbonyloctyl(β-D-galactopyranosyl)-(1-4)--O--(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)--O--(β-D-galactopyranosyl)-(1-4)--O--[β-L-fucopyranosyl-(1-3)--0]-2-acetamido-2-deoxy-β-D-glucopyranoside

Compound 20 (14.0 mg), UDP-Gal (25 mg), βGlcNAc (1-4)galactosyltransferase (14.5 U, Sigma) were incubated for 48 hours in thebuffer described above (3.2 mL). Isolation and purification providedcompound 21 (13.2 mg). ¹ H-n.m.r. data is included in Table II.

C. Synthesis of Compound 22--8-Methoxycarbonyloctyl(5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)--O--(β-D-galactopyranosyl)-(1-4)--O--(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)--O--(β-D-galacto-pyranosyl)-(1-4)--O--[α-L-fucopyranosyl-(1-3)--O]-2-acetamido-2-deoxy-β-D-glucopyranoside

Compound 22 was synthesized from compound 21 as indicated above⁶⁵.

                  TABLE II                                                        ______________________________________                                        .sup.1 H-n.m.r. Structural Parameters                                         Sugar   Hydro-                                                                Unit    gen     19.sup.a,b                                                                              20.sup.a,b                                                                           21.sup.a,b                                                                           22.sup.a,b                            ______________________________________                                        βGlcNAc                                                                          1 (d)   4.50.sup.c (7.5)                                                                        4.52 (7.5)                                                                           4.52 (8.0)                                                                           4.53 (8.0)                            (A)                                                                           βGal (B)                                                                         1 (d)   4.45.sup.c (8.0)                                                                        4.43 (7.0)                                                                           4.43 (7.5)                                                                           4.43 (8.0)                                    4 (d)   4.15 (3.0)                                                                              4.09 (3.5)                                                                           4.10 (3.2)                                                                           4.10 (3.0)                            βGlcNAc                                                                          1 (d)   4.66 (8.5)                                                                              4.67 (8.5)                                                                           4.70 (7.8)                                                                           4.69 (8.0)                            (C)                                                                           βGal (D)                                                                         1 (d)                    4.48 (7.8)                                                                           4.46 (7.7)                                    3 (d)                                                                 αFuc                                                                            1 (d)             5.09 (4.0)                                                                           5.10 (3.8)                                                                           5.09 (3.8)                                    5 (q)             4.81 (6.5)                                                                           4.81 (6.5)                                                                           4.81 (6.5)                                    6 (d)             1.14   1.15   1.15                                  αNeu5Ac                                                                         3.sub.ax                        2.76 (4.5,                                    (dd)                            13.0) 1.79                                    3.sub.eq (t)                    (12.0)                                NHAc    s       2.02, 2.01                                                                              2.02, 2.01                                                                           2.03, 2.02                                                                           2.02                                                                          (three)                               CH.sub.2 CO.sub.2                                                                     t       2.38 (7.5)                                                                              2.38 (7.5)                                                                           2.38 (7.5)                                                                           2.38 (7.5)                            CO.sub.2 CH.sub.3                                                                     s       3.68      3.69   3.69   3.69                                  ______________________________________                                         .sup.a 9, 10, 11 and 12 show multiplets around 1.49-1.63 (4H) and 1.30        (8H): methylenes                                                              .sup.b J in Hz                                                                .sup.c interchangeable                                                   

C. IMMUNOSUPPRESSIVE PROPERTIES

Examples 9 and 10 illustrate the immunosuppressive properties ofhexasaccharide glycoside 5a.

Example 9 Inhibition of DTH Inflammatory Response

DTH inflammatory responses were measured using the mouse footpadswelling assay as described by Smith and Ziola³¹. Briefly, groups ofBalb/c mice were immunized with 10 μg of the L111 S-Layer protein, abacterial surface protein³² from Clostridium thermohydrosulfurum L111-69which has been shown to induce a strong inflammatory DTH response. Sevendays later, each group of mice was footpad-challenged with 10 μg ofL-111 S-Layer protein. The resulting inflammatory footpad swelling wasmeasured with a Mitutoyo Engineering micrometer 24 hours afterchallenge.

To assess the effect of hexasaccharide glycoside 5a on the inflammatoryDTH response, groups of mice received 100 μg of this compound, injectedinto the tail vein, 5 hours after challenge. Control groups received 100μL of phosphate-buffered saline (PBS). The results of this experimentare shown in Table III below. In this table, smaller increases infootpad swelling, as compared to control, evidence the fact that thetested compound possesses immunosuppressive properties in that itreduces the degree of footpad swelling in response to an antigen.

                  TABLE III                                                       ______________________________________                                                          INCREASE IN FOOTPAD                                         COMPOUND TESTED   SWELLING (mm-1)                                             ______________________________________                                        Control           3.3                                                         Hexasaccharide Glycoside 5a                                                                     1.5                                                         ______________________________________                                    

The above results indicate that mice injected with hexasaccharideglycoside 5a had less than 50% of the footpad swelling as compared tothe control mice.

Example 10 Persistence of Suppression of the DTH Inflammatory Responseat 11 Weeks After Challenge

i. The identical groups of mice treated with hexasaccharide glycoside 5ain Example 7 were rechallenged with L111 S-Layer protein 11 weeks afterprimary immunization. Mice treated with the PBS control responded withthe usual degree of footpad swelling whereas mice treated withhexasaccharide glycoside 5a showed a reduction in footpad swelling ofabout 40%, i.e., the mice treated with hexasaccharide glycoside 5aexhibited only about 60% of the footpad swelling exhibited in micetreated with PBS.

This anti-inflammatory effect of hexasaccharide glycosides 5a, given 5hours after the first challenge (one week after primary immunization),had somewhat weakened eleven weeks after primary immunization butnevertheless provided for a significant reduction in inflammation ascompared to PBS treated controls.

In addition to providing suppression of cell-mediated immune responses,the above data demonstrate that treatment with a hexasaccharideglycoside as per this invention also imparts tolerance to additionalchallenges from the same antigen.

Example 11 Effect Hexasaccharide Glycoside 5a has on ELAM-1 DependentCell Adhesion to Activated Vascular Endothelium

This example examines whether hexasaccharide 5a could inhibit ELAM-1dependent cell adhesion to activated vascular endothelium. Specifically,an vitro cell binding assay was preformed as described by Lowe et al³³.Briefly, human umbilical vein endothelial cells (HUVECs purchased fromCell Systems, Seattle, Wash., USA) were stimulated with TNFα (10 ng/ml)to express ELAM-1. Human tumor cell lines, U937 or HL60, which have beenshown to bind to HUVECs, in an ELAM-1 dependent manner were used tomeasure the effect that hexasaccharide glycoside 5a has on the ELAM-1dependent binding to the HUVEC. The results of this example demonstratethat hexasaccharide glycoside 5a inhibits ELAM-1 dependent binding tothe HUVECs.

The data in Examples 9 and 10 above establish the effectiveness of thehexasaccharide glycosides described herein in treating immune responsesto an antigen and in inducing tolerance to the antigen in a mammal(mice). In view of the fact that the immune system of mice is a goodmodel for the human immune system, such hexasaccharide glycosides willalso be effective in treating human immune responses. This is borne outby the fact that Example 11 establishes that hexasaccharide glycoside 5ainhibits ELAM-1 dependent binding to the HUVEC.

By following the procedures set forth in the above examples,hexasaccharide glycosides of formula I and IV above could be used tosuppress a cell-mediated immune response to an antigen by meresubstitution for hexasaccharide glycoside 5a described in theseexamples.

The compounds defined by formula II, III, V and VI are useful at leastas intermediates in the preparation of compounds I and IV. Similarly,the transfer of L-fucose via a (1-3)fucosyltransferase can employ acompatible analog of L-fucose which is recognized by the transferase andprovides for products wherein Y and Y' are compatible analogs ofL-fucose.

What is claim is:
 1. A method for preparation of a compound of theformula I: ##STR12## wherein R is hydrogen, a saccharide, anoligosaccharide or an aglycon group having at least one carbon atom, Yis L-fucose or a compatible analogue of L-fucose, and Z is sialic acidor a compatible analogue of sialic acid, which method comprises(a)fucosylating a compound of the formula II ##STR13## wherein R is asdefined above and X is a removable blocking group with anα(1-3)fucosyltransferase in the presence of a compatible GDP derivativeof L-fucose or an analogue thereof under conditions wherein the L-fucoseis transferred to the 3-position of the N-acetylglucosamine unit at thereducing sugar terminus so as to form a monofucosylated derivative ofthe formula III: ##STR14## wherein X, Y and R are as defined above; (b)removing the removable blocking group from the compound formed in (a)above; and (c) sialylating the compound formed in (b) above with sialicacid or a compatible analogue of sialic acid in the presence of acompatible CMP-derivative of sialic acid or an analogue thereof using anα(2-3)-sialyltransferase under conditions wherein the sialic acid oranalogue thereof is transferred to the 3 position of the theretoforeterminal galactose unit so as to form the compound of formula I.
 2. Amethod according to claim 1 wherein R is selected from the groupconsisting of --(A)--Z' wherein A represents a bond, an alkylene groupof from 2 to 10 carbon atoms, and a moiety of the form --(CH₂ -CR₂G)_(n) -- wherein n is an integer equal to 1 to 5; R₂ is selected fromthe group consisting of hydrogen, methyl, and ethyl; and G is selectedfrom the group consisting of hydrogen, oxygen, sulphur, nitrogen, phenyland phenyl substituted with 1 to 3 substituents selected from the groupconsisting of amine, hydroxyl, halo, alkyl of from 1 to 4 carbon atomsand alkoxy of from 1 to 4 carbon atoms; and Z' is selected from thegroup consisting of hydrogen, methyl and, when G is not oxygen, sulphuror nitrogen and A is not a bond, then Z' is also selected from the groupconsisting of --OH, --SH, --NH₂, --NHR₃, --N(R₃)₂, -- C(O)OH, --C(O)OR₃,--C(O)NH--NH₂, --C(O)NH₂, --C(O)NHR₃, --C(O)N(R₃)₂, and --OR₄ whereineach R₃ is independently alkyl of from 1 to 4 carbon atoms and R₄ is analkenyl group of from 3 to 10 carbon atoms.
 3. A method according toclaim 2 wherein the blocking group is selected from the group consistingof sialic acid and benzyl.
 4. A method for preparation of a compound ofthe formula IV: ##STR15## wherein R is hydrogen, a saccharide, anoligosaccharide, or an aglycon group having at least one carbon atom, Y'is L-fucose or a compatible analogue of L-fucose, and Z is sialic acidor a compatible analogue of sialic acid, which method comprises(a)sialylating a compound of the formula V ##STR16## wherein R is asdefined above and X' is a removable blocking group with asialyltransferase in the presence of a compatible CMP derivative ofsialic acid or a compatible analogue of sialic acid using anα(2-3)sialyltransferase under conditions wherein the sialic acid oranalogue thereof is transferred to the 3 position of the theretoforeterminal galactose unit; and (b) fucosylating the compound prepared in(a) above with an α(1-3)fucosyltransferase in the presence of acompatible GDP derivative of L-fucose or an analogue thereof underconditions wherein L-fucose is transferred to the 3-position of thenon-reducing sugar N-acetylglucosamine unit so as to form amonofucosylated derivative of the formula VI: ##STR17## wherein X', Y'and R are as defined above; and (c) removing the removable blockinggroup from the compound formed in (b) above so as to form a compound offormula IV with the proviso that X' is not sialic acid.
 5. A methodaccording to claim 4 wherein R is selected from the group consisting of--(A)--Z' wherein A represents a bond, an alkylene group of from 2 to 10carbon atoms, and a moiety of the form --(CH₂ --CR₂ G)_(n) -- wherein nis an integer equal to 1 to 5; R₂ is selected from the group consistingof hydrogen, methyl, and ethyl; and G is selected from the groupconsisting of hydrogen, oxygen, sulphur, nitrogen, phenyl and phenylsubstituted with 1 to 3 substituents selected from the group consistingof amine, hydroxyl, halo, alkyl of from 1 to 4 carbon atoms and alkoxyof from 1 to 4 carbon atoms; and Z' is selected from the groupconsisting of hydrogen, methyl and, when G is not oxygen, sulphur ornitrogen and A is not a bond, then Z' is also selected from the groupconsisting of --OH, --SH, --NH₂, --NHR₃, --N(R₃)₂, --C(O)OH, --C(O)OR₃,--C(O)NH--NH₂, --C(O)NH₂, --C(O)NHR₃, --C(O)N(R₃)₂, and --OR₄ whereineach R₃ is independently alkyl of from 1 to 4 carbon atoms and R₄ is analkenyl group of from 3 to 10 carbon atoms.
 6. A method according toclaim 5 wherein the blocking group is a benzyl group.
 7. The methodaccording to claim 1 wherein fucosylation procedure (a) and sialylationprocedure (c) are conducted at a temperature of from 25° to 45° C.; fora period of time of from 12 hours to 4 days; and at a pH of from about6.5 to 7.5.
 8. The method according to claim 4 wherein sialylationprocedure (a) and fucosylation procedure (c) are conducted at atemperature of from 25° to 45° C.; for a period of time of from 12 hoursto 4 days; and at a pH of from about 6.5 to 7.5.
 9. A method for thepreparation of a compound of formula I: ##STR18## wherein R is hydrogen,a saccharide, an oligosaccharide, or an aglycon group having at leastone carbon atom, Y is L-fucose or a compatible analogue of L-fucose andZ is sialic acid or a compatible analogue of sialic acid, which methodcomprises:(a) fucosylating a compound of the formulaβGlcNAc(1→3)βGal(1→4)βGlcNAc-OR with an α(1-3)fucosyltransferase in thepresence of a GDP derivative of L-fucose under conditions sufficient toform the compound: βGlcNAc(1→3)βGal(1→4)βGlcNAc-OR; (b) galactosylatingthe compound prepared in (a) above with a suitable galactosyltransferasein the presence of uridine 5' (galactopyranosyl)-diphosphate underconditions sufficient to form the compound:βGal(1→4)βGlcNAc(1→3)βGal(1→4)βGlcNAc-OR; (c) sialylating the compoundformed in (b) above with a sialyltransferase in the presence of acompatible CMP derivative of sialic acid or an analogue of sialic acidusing an α(2-3)sialyltransferase under conditions wherein the sialicacid or analogue thereof is transferred to the non-reducing sugar so asto form the compound of formula I above.
 10. The method according toclaim 9 wherein the compound of the formulaβGlcNAc(1→3)βGal(1→4)βGlcNAc-OR is prepared by:galactosylating acompound of the formula GlcNAc-OR with a suitable galactosyltransferasein the presence of uridine 5' (galactopyranosyl)-diphosphate underconditions sufficient to form βGal(1→4)GlcNAc-OR; andN-acetylglucosaminylating the compound produced above with a suitableN-acetylglucosaminyltransferase in the presence of uridine 5'N-(acetylglucosamine)-diphosphate under conditions sufficient to formthe compound βGlcNAc(1→3)βGal(1→4)βGlcNAc-OR.